Hyperinsulinemic Euglycemic Clamp Protocol
Title
A Phase 2A, Double-blind, Placebo-controlled, Randomized
Study to Evaluate the Safety and Efficacy of TRC150094 in
Increasing Insulin Sensitivity in Male patients with increased
Cardiometabolic Risk
Study Number: CT/P015/CMR/2010/02_01
Protocol Version: 3.0
Protocol Date: 18/11/2011
CTRI Number: REFCTRI-2010 002973
Investigational
Medicinal Product:
TRC150094
Indication: Cardiometabolic Risk associated with non-traditional risk
factors
Clinical Phase: Phase 2A
Sponsor: Torrent Pharmaceuticals Limited
Torrent Research Centre
Village Bhat
Dist Gandhinagar
Gujarat, India
Tel: +91 79 23969100
Fax: +91 79 23969135
Principal
Investigators:
Prof. E.S.G Stroes (AMC, The Netherlands)
Dr. Dharmesh Domadia (Veeda CR, India)
The information contained herein is strictly confidential and must not be disclosed, copied,
submitted for publication, or used for any purpose without the sponsor’s prior written
authorisation.
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INVESTIGATOR SIGNATURE PAGE
A Phase 2A, Double-blind, Placebo-controlled, Randomized
Study to Evaluate the Safety and Efficacy of TRC150094 in
Increasing Insulin Sensitivity in Male patients with increased
Cardiometabolic Risk
I confirm that I have read and I understand this protocol and other appropriate related
documentation, including the Investigator’s Brochure for TRC150094. I agree that the
available pre-clinical information on the investigational product is adequate to support the
proposed clinical trial. I agree to perform this study in accordance with this protocol, the
ethical principles that have their origin in the Declaration of Helsinki, the International
Conference on Harmonisation (ICH) guideline on Good Clinical Practice (GCP), and the
applicable regulatory requirement(s). I will also appropriately direct and assist the personnel
at the trial site who will be involved in the conduct of the study.
Prof. E.S.G Stroes
Department of Vascular Medicine, AMC
Amsterdam, The Netherlands
Date
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SPONSOR APPROVAL SIGNATURE PAGE
A Phase 2A, Double-blind, Placebo-controlled, Randomized
Study to Evaluate the Safety and Efficacy of TRC150094 in
Increasing Insulin Sensitivity in Male patients with increased
Cardiometabolic Risk
Approved by:
Dr. Chaitanya Dutt
Director, R&D
Torrent Pharmaceuticals Limited
Torrent Research Centre
Village Bhat
Dist Gandhinagar 382 428
Gujarat
India
Date
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SPONSOR CONTACT DETAILS*
Study Monitor: Dr. Lalit Lakhwani
Scientist I, Clinical Research
Tel: +91 79 23969100 Ext. 194
Mobile: +91 9227400443
Email: [email protected]
Fax: +91 79 23969135
Project Manager: Mr. Kashyap Avashia
AGM, TCM-Discovery
Tel: +91 79 23969100 Ext. 566
Mobile: +91 9727704562
Email: [email protected]
Fax: +91 79 23969135
Medical Experts:
Prof. E.S.G. Stroes MD, PhD
Department of Vascular Medicine, AMC
Tel: +31 20 566 5978
Email- [email protected]
Dr. Chaitanya Dutt
Director, R&D
Tel: +91 79 23969100 Ext. 101
Mobile: +91 9825606901
Email: [email protected]
Fax: +91 79 23969135
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24-Hour Emergency
Contacts/ SAE Reporting:
The 24 hour Emergency Contacts will be contacted as per the order mentioned
below:
Dr. Lalit Lakhwani
Scientist I, Clinical Research
Tel: +91 79 23969100 Ext. 194
Mobile: +91 9227400443
Email: [email protected]
Fax: +91 79 23969135
Dr. KumarPrafull Chandra
AGM, Clinical Research
Tel: +91 79 23969100 Ext. 193
Mobile: +91 9909004214
Email: [email protected]
Fax: +91 79 23969135
Dr. Ambrish Srivastava
General Manager, Clinical Research
Tel: +91 79 23969100 Ext. 181
Mobile: +91 9879107948
Email: [email protected]
Fax: +91 79 23969135
* contact address for all above:
Torrent Research Centre
Village Bhat, Dist Gandhinagar 382 428
Gujarat, INDIA
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CONTACT DETAILS FOR PERSONNEL AT STUDY SITES
Academic Medical Centre, Amsterdam
Role Name Telephone Email
Principal
Investigator
Prof. E.S.G.
Stroes MD, PhD
Department of
Vascular
Medicine, AMC
+31 20 566 5978 [email protected]
Independent
physician M.D.trip MD,
PhD
Department of
Vascular
Medicine AMC
+31 20 566 65882 [email protected]
Project
Manager H. Suwier
ClinResearch
Consulting
BV.BVBA
0032-50673524
Mobile
+32475687662
Veeda Clinical Research, Ahmedabad
Role Name Telephone Email
Principal
Investigator Dr. Dharmesh
Domadia
079-30013060
Mobile: +91
9879590828
Co-investigator Dr. Hardik
Dave
079-30013010 (Ext 245) [email protected]
Project
Manager Mr. Prakash
Patel
079-30013010 (Ext 244)
Mobile:+91 9909930321
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DETAILS OF STUDY FACILITIES AND PERSONNEL
A. ACADEMIC MEDICAL CENTRE, AMSTERDAM
Principal investigator
E.S.G. Stroes MD, PhD
Department of Vascular Medicine, AMC
Meibergdreef 9, room F4-275
1100 DD Amsterdam, The Netherlands
E-mail:[email protected]
Tel: +31 20 566 5978, pager nr: 8158287
Independent physician M.D.trip MD, PhD
Department of Vascular Medicine AMC
Meibergdreef 9, room F4-109
1100 DD Amsterdam, The Netherlands
E-mail: [email protected]
Tel: +31 20 566 65882, pager nr: 8159520
Laboratory sites W. Schornagel
LAKC, AMC
Meibergdreef 9, room B1-238
1100 DD Amsterdam, The Netherlands
E-mail: [email protected]
Tel: +31 20 5665866
M.T.Ackermans
Lab. Spec. Endocrinology, AMC
Meibergdreef 9, room F2-131.3
1100 DD Amsterdam, The Netherlands
E-mail: [email protected]
Tel: +31 20 5665924 pager nr: 8165924
G.M.Dallinga-thie
Lab. Exp. Vascular medicine
Meibergdreef 9, room K1-262
1100 DD Amsterdam, The Netherlands
E-mail: [email protected]
Tel: +31 20 5665158
Pharmacy E.M.Kemper, PhD
Pharmacy, AMC
Meibergdreef 9, room E0B-100
1100 DD Amsterdam, The Netherlands
E-mail: [email protected]
Tel: +31 20 5667955
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B. VEEDA CLINICAL RESEARCH
Principal Investigator Dr. Dharmesh Domadia
Veeda Clinical Research, Ahmedabad
Co-investigator Dr. Hardik Dave
Project manager Mr. Prakash Patel
Pharmacy Mr. Pankaj Sojitra
Clinical Laboratory Tests
(Safety)
Supratech Micropath Laboratory & Research
Institute
‘KEDAR’ Opp. Krupa Petrol Pump
Nr. Parimal Garden
Ahmedabad – 380 006, India.
Phone No.: +91-79-2640 8181
+91-79-2640 8182
Fax No.: +91-79-2640 9292
Radiological Tests Shachi Digital X-Ray, Sonography and Color
Doppler Clinic
F-2, 3, Balaji centre, Opp. Gurukul, Drive-in road,
Memnagar, Ahmedabad-380 052, India.
Phone: +91-79-2749 1622
Emergency Care Hospital Sterling Hospital
Memnagar
Ahmedabad – 380 052, India
Phone: +91-79-2748 1415 / 5767
C. TORRENT RESEARCH CENTRE
SIRT Expression analysis Dr Shailesh Deshpande
Cell and molecular Biology Lab,
Torrent Pharmaceuticals Limited
Torrent Research Centre
Dist Gandhinagar 382 428
Gujarat,India
Phone: +91-79-23969100 Ext. 715
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1. TABLE OF CONTENTS
Investigator Signature Page ...........................................................................................2
Sponsor Approval Signature Page .................................................................................3
Sponsor Contact Details.................................................................................................4
Contact Details for Personnel at Study Site...................................................................6
Details of Study Facilities and Personnel ......................................................................7
1. TABLE OF CONTENTS.......................................................................................9
2. SYNOPSIS...........................................................................................................14
3. INTRODUCTION ...............................................................................................19
3.1 Rationale for Current Study.................................................................25
4. OBJECTIVES......................................................................................................26
4.1 Primary Objective................................................................................26
4.2 Secondary Objectives ..........................................................................26
5. STUDY DESIGN.................................................................................................27
5.1 Overview .............................................................................................27
5.2 Rationale for Study Design, Control, Doses and Study Population ....28
6. STUDY POPULATION AND Treatment ...........................................................29
6.1 Number and Description of Subjects...................................................29
6.2 Inclusion Criteria .................................................................................29
6.3 Exclusion Criteria ................................................................................30
6.4 Sample Size Calculation......................................................................31
6.5 Investigational product ........................................................................31
6.6 Prior and Concomitant Medication......................................................31
6.7 Restrictions on Subjects.......................................................................32
6.7.1 Diet.......................................................................................................32
6.7.2 Alcohol, caffeine, grapefruit...............................................................32
6.7.3 Exercise ...............................................................................................32
7. INVESTIGATIONAL PRODUCT Administration ............................................33
7.1 Description of investigational products ...............................................33
7.2 Summary of pharmaceutical, nonclinical and clinical information of
TRC150094 33
7.2.1 Physical, Chemical & Pharmaceutical summary ..............................33
7.2.2 Nonclinical Studies .............................................................................33
7.2.3 Available Clinical Data.......................................................................36
7.2.4 Summary of potential risks and benefits ............................................39
7.2.5 Description and justification of route of administration and dosage39
7.3 Dose Administration............................................................................40
7.4 Supply, Identification and Storage ......................................................40
7.5 Treatment Compliance ........................................................................41
7.6 Treatment of Overdose ........................................................................42
7.7 Accountability .....................................................................................42
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7.8 Dispensing of Investigational Product.................................................42
8. METHODS ..........................................................................................................43
8.1 Study parameters/endpoints.................................................................43
8.1.1 Main study parameter/endpoint..........................................................43
8.1.2 Secondary study parameters/endpoints ..............................................43
8.2 Randomisation and treatment allocation .............................................43
8.2.1 Preparation of randomisation code ....................................................43
8.2.2 Breaking the randomisation code.......................................................43
8.3 Blinding ...............................................................................................44
8.4 Study Procedures .................................................................................44
8.4.1 Hyperinsulinemic Euglycemic Clamp................................................44
8.4.2 Quantification of Hepatic Fat. ..........................................................46
8.4.3 Silent Information Regulator T (SIRT) expression study: ............47
8.4.4 Measurement of Sagittal Abdominal diameter ...............................47
8.5 Study Visits..........................................................................................47
8.5.1 . Clinical laboratory tests ....................................................................51
8.5.2 Vital signs ............................................................................................52
8.5.3 12-Lead ECGs .....................................................................................52
8.5.4 Physical examinations ........................................................................52
8.5.5 Chest X ray ..........................................................................................52
8.5.6 Laboratory investigations and bioanalysis of blood samples ............52
8.5.7 Bioanalysis ..........................................................................................54
8.5.8 Sample retention / destruction............................................................54
8.6 Appropriateness of Measurements ......................................................54
8.7 Total Volume of Blood........................................................................54
8.8 Withdrawal Criteria .............................................................................55
8.9 Withdrawal Procedures........................................................................55
8.10 Replacement of Dropout/Withdrawals ................................................55
8.11 Follow up of subjects withdrawn from treatment................................56
8.12 Study Termination ...............................................................................56
9. SAFETY Reporting .............................................................................................56
9.1 Safety and tolerability assessments and reporting...............................56
9.1.1 Safety Monitoring ...............................................................................56
9.1.2 Section 10 WMO event........................................................................56
9.1.3 Adverse events and serious adverse events.........................................57
9.1.4 Reporting of adverse event..................................................................57
9.1.5 Procedures for reporting SAEs...........................................................58
9.1.6 Emergency procedures........................................................................59
9.1.7 Suspected unexpected serious adverse reactions (SUSAR)...............59
9.1.8 Annual safety report............................................................................59
9.1.9 Follow-up of adverse events ...............................................................60
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10. DATA MANAGEMENT AND STATISTICAL ANALYSIS............................61
10.1 Sample Size Calculation......................................................................61
10.2 Data Handling......................................................................................61
10.3 Statistical Analysis ..............................................................................61
10.3.1 Descriptive statistics ............................................................................61
10.3.2 Univariate analysis..............................................................................61
10.4 Safety and Tolerability Analysis .........................................................62
11. ETHICAL AND REGULATORY CONSIDERATIONS...................................62
11.1 Regulation statement ...........................................................................62
11.2 Recruitment and consent .....................................................................62
11.3 Benefits and risks assessment, group relatedness................................63
11.4 Compensation for study related illness/injury .....................................64
12. ADMINISTRATIVE ASPECTS AND PUBLICATION....................................64
12.1 Handling and storage of data and documents ......................................64
12.2 Amendments ........................................................................................65
12.3 Annual progress report ........................................................................66
12.4 End of study report ..............................................................................66
12.5 Public disclosure and publication policy .............................................66
13. QUALITY CONTROL AND QUALITY ASSURANCE...................................67
14. REFERENCES ....................................................................................................74
LIST OF TABLES
Table 1: Summary of Blood Volumes for Each Subject..............................................54
LIST OF FIGURES
Figure 1: Pathologies originating form visceral adiposity and Insulin resistance……19
Figure 2: Concept of cardiometabolic risk…………………………………………...20
LIST OF APPENDICES
Appendix A: Clinical laboratory tests………………………………………………..67
Appendix B: Study visits……………………………………………………………..68
Appendix C: Flow chart of clamp procedure………………………………………...71
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LIST OF ABBREVIATIONS
AE Adverse Event
ACE Angiotensin Converting Enzyme
ALT Alanine Transaminase
Apo B Apolipoprotein B
aPTT Activated Partial Thromboplastin Time
AST Aspartate Transaminase
ASCVD Atherosclerotic Cardiovascular Disease
AUC Area Under the Curve
Od Once daily
BMI Body Mass Index
BUN Blood Urea Nitrogen
Cmax Maximum observed plasma drug concentration
CVD Cardiovascular Disease
CVS Cardiovascular System
CHD Coronary Heart Disease
CMR Cardiometabolic Risk
CNS Central Nervous System
CRF Case Report Form
CRO Clinical research organisation
CRU Clinical Research Unit
CTA Clinical Trial Application
CYPs Cytochrome P
DIO Diet Induced Obesity
ECG Electrocardiogram
EP Endogenous Production
eGFR Estimated Glomerular Filtration Rate
FFA Free Fatty Acid
fT3 Free T3
fT4 Free T4
GCP Good Clinical Practice
GGT Gamma Glutaryl Transferase
GI GastroIntestinal
GMP Good Manufacturing Practice
GST Glutathione S-Transferase
HBsAg Hepatitis B surface Antigen
HDL High Density Lipoprotein
HED Human Equivalent Dose
HIV Human immunodeficiency virus
Hs CRP Highly sensitive C-Reactive Protein
ICF Informed consent form
ICH International Conference on Harmonisation
IEC Independent Ethics Committee
IL6 Interleukin 6
IMPD Investigational Medicinal Product Dossier
IR Insulin Resistance
IRB Institutional Review Board
LC/MS/MS Liquid Chromatography/Mass Spectrometry/Mass Spectrometry
LDL Low Density Lipoprotein
Lp Lipoprotein
LVP Left Ventricular Pressure
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MedDRA Medical Dictionary for Regulatory Activities
MDRD Modification of Diet in Renal Disease
MAD Multiple Ascending Dose
MCP-1 Monocyte Chemotactic Protein 1
MI Myocardial Infarction
IEC Medical Research Ethics Committee
MRS Magnetic Resonance Spectroscopy
RMR Resting Metabolic Rate
MABEL Minimal Anticipated Biological Effect Level
NOAEL No-Observed-Adverse-Effect Level
NAFLD Non-alcoholic Fatty Liver Disease
NEFA Non-esterified Fatty Acid
Od Once daily
PAI-1 Plasminogen Activator Inhibitor
PBMNC Peripheral blood mono-nuclear cells
PCOD Polycystic Ovary Disease
PCR Polymerase Chain Reaction
PK Pharmacokinetic
3-OHB 3-hydroxybutyrate
SCD-1 Stearoyl-CoA desaturase-1
PPAR- γ Peroxisome Proliferator Activated Receptor- γ
QTc Corrected QT
SAD Single Ascending Dose
SAE Serious Adverse Event
SIRT Silent Information Regulator T
SUSAR Suspected Unexpected Serious Adverse Reaction
T2 Diiodothyronine
T3 Triiodothyronine
T4 Thyroxine
TNF Tumour Necrosis Factor
TR Thyroid Receptor
TSH Thyroid Stimulating Hormone
ULN Upper Limit of Normal
VAT Visceral Adipose Tissue
VLDL Very Low Density Lipoprotein
WHO World Health Organization
WMO Wet Medisch-wetenschappelijk Onderzoek met Mensen (Medical
Research Involving Human Subjects Act)
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2. SYNOPSIS
Sponsor:
Torrent Pharmaceuticals Limited, Torrent Research Centre, Village Bhat,
Dist. Gandhinagar, Gujarat, India.
Study number:
CT/P015/CMR/2010/02_01
Investigational medicinal product:
TRC150094
CTRI number: REFCTRI-2010 002973
Title of study:
A Phase 2A, Double-blind, Placebo-controlled, Randomized Study to Evaluate the
Safety and Efficacy of TRC150094 in Increasing Insulin Sensitivity in Male patients
with increased Cardiometabolic Risk.
Principal Investigators:
1. Prof. E.S.G Stroes at Academic Medical Centre, Amsterdam, The Netherlands
2. Dr. Dharmesh Domadia at Veeda Clinical Research, Ahmedabad, India
Study centres:
1. Academic Medical Centre, Amsterdam, The Netherlands
2. Veeda Clinical Research, Ahmedabad, India
Planned study period:
6 months
Clinical Phase:
Phase 2A
Objectives:
Primary: To determine the safety and efficacy (in increasing insulin sensitivity)
of TRC150094 once daily dosing for 4 weeks in male patients with
increased cardiometabolic risk.
Secondary:
• To evaluate the effect of TRC150094 on hepatic fat and metabolic
parameters.
• To evaluate the ethnic differences for effect of TRC150094 on Insulin
sensitivity parameters
Study Design:
This is a Phase 2A, two-centre, double-blind, randomized, placebo-controlled,
multiple-dose, parallel study. Each subject will attend the study centre for 1
screening visit, 2 study visits (1 baseline and 1 end of treatment), 1 intermediate
safety visit and 1 post-study follow-up visit (Total 5 visits). Total 40 subjects will
take part in the study; 20 subjects will be enrolled in Ahmedabad, India and 20 will
be enrolled in Amsterdam, Netherlands. Subjects will be randomized 1:1 for active
treatment versus placebo. Dosing will take place for 28 days (Days 1−28).
Inclusion Criteria
Subjects will be considered eligible for entry in the study if they meet all
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of the following criteria.
1. Adult male
2. Age range 30−65 years at screening
3. Caucasian or Indian ethnicity
4. Waist circumference ≥ 102 cm for Caucasians and ≥ 90 cm for
Indians at screening.
5. Fasting Serum Insulin ≥ 10 mU/ml at screening
6. Blood Pressure ≥ 130/85 mmHg at screening (or patients taking
medication for hypertension)
7. Stable weight during 3 months prior to the study (assessed through
medical history of the patient)
8. Drug naive diabetic patients* or patients with impaired fasting
glucose i.e > 100 mg/dl or 5.5 mmol/l and < 200 mg/dl or 11.0
mmol/l Diabetic patients who were taking metformin and have
undergone washout for at least 4 weeks before Day 0 and are
currently on life style modification as a treatment for diabetes will
also be allowed in the study
9. Willingness to give written informed consent (prior to any study-
related procedures being performed) and ability to adhere to the study
restrictions and assessments schedule.
* Diabetic patient is defined as a patient with a documented history of type II
DM or a documented history of a fasting glucose > 200mg/dl or 11.0 mmol/l or
2x fasting glucose > 126 mg/dl or 6.9 mmol/l (2x =recorded twice).
Exclusion Criteria
Subjects will not be considered eligible for entry in the study if they meet one or
more of the following criteria.
1. Medical history, physical examination, vital signs, clinical laboratory
tests, 12-lead ECG and Chest X ray (to exclude tuberculosis in India
only) with any significant abnormalities, in the opinion of the
investigator.
2. Subjects with any known somatic illness, including neoplasm,
endocrine disorder such as cushing’s disease, PCOD and uncontrolled
hypothyroidism, neurologic disorder, active infection, or recent
surgical procedure within 3 months of the study initiation.
3. Subject currently using medication, which can influence glucose or
FFA metabolism such as fibrates, niacin, ACE inhibitors (subjects are
not excluded in case of use of a stable dose for at least 6 weeks prior
to baseline measurement, taking (ACE) inhibitors (ACE-I) or
angiotensin-receptor blockers (ARBs)), PPAR agonists, omega 3 fatty
acids.
4. eGFR < 60 mL/min/1.73m2 at screening as evaluated by Modification
of Diet in Renal Disease (MDRD) method.
5. History of angina, Myocardial Infarction (MI) or stroke since last 6
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months.
6. Hypertension with SBP/DBP ≥160/100 mmHg at screening.
7. ALT or AST ≥ ULN*3 at screening
8. History or presence of malignancy.
9. History of recreational drug use within the last 30 days, or regular
consumption of greater than 2 units of alcohol/day.
10. History of allergy to the test drug or any drug chemically similar to the
drug under investigation.
11. Seropositive for Hepatitis B, Hepatitis C or HIV.
12. Subjects suffering from any psychiatric (acute or chronic) illness.
13. Intake of any medication except those permitted in this study (see
Section 6.6).
14. Intake of any investigational drug in the period within 3 months prior
to the first dose of study drug.
15. History of significant blood loss due to any reason, including blood
donation, in the 12 weeks prior to the first dose of study drug; or the
total blood loss in the last 3 months, including for this study, exceeds
450 mL.
16. History of any bleeding disorder.
17. Existence of any surgical or medical condition which, in the judgment
of the principal investigator, might interfere with the absorption,
distribution, metabolism or excretion of the study drug or might be
likely to compromise the safety of the subject.
18. Inability to communicate or co-operate with the investigator because
of language problems, poor mental development or impaired cerebral
function.
19. Inability to comply with study requirements.
20. Positive drugs of abuse test (at screening) and alcohol breath test.
21. Heavy smokers (who are smoking >15 cigarettes or equivalent per
day).
Test product, dose and mode of administration:
TRC150094 tablets, Tentative dose 50 mg (1 x 50 mg tablet)
Mode of administration: oral
Duration of treatment:
Once daily dosing on Days 1−28
Reference therapy, dose and mode of administration:
Placebo tablets for oral administration (identical taste, appearance to TRC150094
tablets)
Criteria for evaluation:
Insulin Sensitivity:
• Rate of Glucose Disposal
• Suppression of Endogenous Glucose Production
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• Suppression of rate of lipolysis
Early efficacy markers that will be explored include:
• Hepatic fat
• Lipid parameters
• Metabolic markers
Methodology
On screening, patients will visit the trial unit after an overnight fast. After informed
consent is obtained, subjects will be screened for eligibility to participate in the
study. Screening will include a careful assessment of the inclusion/exclusion criteria
and collection of blood and urine for screening tests. Subjects will give a full
medical history, and undergo a full physical examination, including height, body
weight, waist circumference and vital sign measurements. A 12-lead
electrocardiogram (ECG) tracing will be obtained to screen for underlying cardiac
disease. All subjects will receive life style instructions (normal diet, ≤ 2 alcohol
consumptions a day, restriction on caffeine and grapefruit intake, no strenuous or
unaccustomed exercise). If a patient is on oral metformin treatment, treatment will be
discontinued until the end of the study. Subjects who meet all entry criteria will be
eligible for randomization on Day 0.
On Day 0 patients will visit the trial unit after an overnight fast. The patients may be
asked to present in CRU on Day -1 if required (for baseline investigations). At Day 0
(and/or Day -1), the following assessments will be performed: Physical examination,
vital signs, height and body weight, waist circumference, clinical laboratory tests,
urine collection (urinalysis), 12-lead ECG, AEs and change in concomitant
medications will be reported, hyperinsulinemic euglycemic clamp (the clamp
procedure is described in Appendix C) will be performed. Liver fat will be measured
by 1H MRS. All patients will get life style instructions.
On day14±1, the patients will visit the trial unit after an overnight fast. The
following assessments will be performed on day 14±1: vital signs, height and body
weight, waist circumference, safety clinical laboratory tests, urine collection
(urinalysis), 12-lead ECG, AEs and change in concomitant medications will be
reported, all patients will get life style instructions.
On Day 28+2, patients will visit the trial unit after an overnight fast. At Day 28, the
following assessments will be performed: physical examination, vital signs, height
and body weight, waist circumference, clinical laboratory tests, urine collection
(urinalysis), 12-lead ECG, AEs and change in concomitant medications will be
reported, hyperinsulinemic euglycemic clamp (the clamp procedure is described in
Appendix C) will be performed, liver fat will be measured by 1H MRS,
discontinuation of study drug, all patients will get life style instructions.
At Day 35-38, patients will visit the trial unit after an overnight fast and the
following assessments will be performed: physical examination, vital signs, height
and body weight, waist circumference, safety clinical laboratory tests, urine
collection (urinalysis), 12-lead ECG, AEs and change in concomitant medications
will be reported, restart of oral metformin treatment (if applicable).
For subjects who are withdrawn, the above assessments should be performed within
8-10 days after the last drug administration.
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Benefit and risk
This study does not have specific advantages for the study subjects. The results of
this study may help researchers learn whether TRC150094 may be beneficial for the
treatment of male subjects with increased cardiometabolic risk associated with non-
traditional risk factors. [6,6-2H2] glucose is glucose labeled with a stable isotope of
hydrogen, which behaves as the natural substrate and has no side effects. [1,1,2,3,3-
2H2] labelled glycerol is glycerol labeled with 5 stable isotope of hydrogen, which
behaves as the natural substrate and has no side effects. Actrapid is fast acting
insulin, a hormone that could induce hypoglycemia. However, it is not in the scope
of this protocol to allow hypoglycemia to occur, since plasma glucose concentration
will be fixed at 5 mmol/l by a variable infusion of glucose 20% guided by frequent
bedside glucose measurements. An overview of the blood samples and blood
volumes taken during the study is provided in Table 1. The total blood volume to be
withdrawn from any individual subject will be 391.2 ml for subjects at Veeda
Clinical Research, India and 361.2 ml at AMC, Netherlands. This amount is not
considered to be of negative influence to the subject’s health. A possible side effect
of clamping, which is very rare and mostly preventable, could be hypoglycaemia.
MR- spectroscopy of the liver will be made which takes about 30 minutes. This
spectroscopy is not considered to be potentially harmful to subjects.
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3. INTRODUCTION
Background
A plethora of studies have shown that visceral adiposity and insulin resistance are the
key underlying factors associated with clustering atherogenic abnormalities which
include a typical atherogenic dyslipidemic state (high triglyceride and apolipoprotein
B concentrations, an increased proportion of small dense LDL particles and a reduced
concentration of HDL-cholesterol with HDL particles also being smaller in size), a
prothrombotic profile, and a state of inflammation. Furthermore, visceral adiposity
and insulin resistance could also contribute to an elevated blood pressure and to
dysglycemia1 eventually leading to Atherogenic Cardiovascular Disease
2,3,4 and Type
2 diabetes5,6
Fig 1 describes the pathologies originating from Visceral adiposity and
Insulin Resistance.
Fig 1: Pathologies originating from Visceral Adiposity and Insulin Resistance
The incidence of visceral adiposity and the related comorbidities are increasing
worldwide, leading to increased burden of cardiovascular risk in general population.
Traditional risk factors as defined by Framingham Heart Study (age, race, gender,
smoking, hypertension, High Cholesterol and family history) do not completely
account for the cardiovascular risk in majority of population. Non-traditional risk
factors (also known as Non-framingham risk factors) such as obesity, insulin
resistance, dyslipidemia, dysglycemia and Hypertension are equally important culprit
for increase in CV risk. Hence Cardiometabolic risk which is the overall risk of
Visceral
Adiposity
Impaired
Fibrinolysis
Dysglycemia Atherogenic
Dyslipidemia
Increased
Blood Pressure
Inflammation
Insulin
Resistance
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cardiovascular disease (CVD) and diabetes resulting from the presence of the non-
traditional risk factors and also of traditional risk has been described as a plausible
target for treatment1. Figure 2 elucidates the concept of Cardiometabolic risk.
Fig 2: Concept of Cardiometabolic Risk
The pathophysiology underlying Cardiometabolic risk is complex and has been only
partially elucidated.
There is considerable evidence supporting the notion that excess abdominal fat is
predictive of insulin resistance and the presence of related metabolic abnormalities.
Studies of the measurement of abdominal adiposity (magnetic resonance imaging and
computed tomography) have commonly reached the conclusion that the amount of
visceral adipose fat not that of subcutaneous abdominal fat, is a quite dominant
correlate of metabolic abnormalities observed in overweight/obese patients
Visceral Adipose tissue (VAT) is not only an energy storage tissue, but also a
metabolically active organ secreting hormones, cytokines and growth factors,
collectively called as adipocytokines. It is believed that anti-atherosclerotic
adipocytokines, such as leptin and adiponectin, and proatherosclerotic cytokines, such
as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, cooperatively regulate
metabolic and cardiovascular homeostasis at local and remote sites. Visceral adiposity
perturbs this homeostasis thus resulting in pro-atherogenic state.7
Visceral adiposity is also associated with a pro-inflammatory state which is also an
etiological factor for the atherosclerotic process. Overall, these pathophysiological
Non-traditional risk factors
1. Insulin resistance
2. Dysglycemia
3. Dyslipidemia
4. Hypertension
5. Visceral obesity
Traditional risk factors
1. Age, male gender
2. Smoking
3. Hypertension
4. Total, HDL Cholesterol
5. Diabetes
6. Others/genetic factors
Cardiometabolic Risk
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processes culminate into increased risk of ASCVD (Atherosclerotic Cardiovascular
Disease).2,3,4
.
Visceral adiposity is an important causative factor for whole body Insulin resistance.
Visceral adiposity leads to increased fatty acid load to liver and muscle forming an
Insulin resistant milieu in liver and muscle tissues.1
Visceral adiposity and associated insulin resistance contributes to exposure of high
concentrations of free fatty acids (due to increased lipolysis) to the liver (through the
portal circulation), impairing several hepatic metabolic processes leading to
hyperinsulinemia (decreased insulin clearance), glucose intolerance (increased hepatic
glucose production), and hypertriglyceridemia (increased VLDL-apolipoprotein B
secretion).1
Intra-hepatic fat thus represents an important contributor to the pathogenesis of
Cardiometabolic risk.
In fact various global studies (eg The Framingham Heart Study) have shown that
adiposity represented by increased intrahepatic fat (IHF), compared with general
adiposity (represented by BMI), would explain more of the variance in cardiovascular
disease (CVD) risk factors.8
Insulin resistance (IR) is another important component of cardiometabolic risk and
consists of two important features; peripheral and hepatic insulin sensitivity.
Peripheral insulin resistance leads to decreased insulin stimulated glucose uptake in
skeletal muscle and adipocytes. Hepatic IR increases plasma glucose levels by
decreasing insulin mediated suppression of glucose production. The physiological
mechanisms underlying insulin resistance are complex and not fully elucidated.
Amongst others, an impairment of the skeletal muscle metabolic energy transduction
pathways (mitochondrial dysfunction) has been suggested to be casually related to
insulin resistance. Mitochondrial fatty acid overload and incomplete fatty acid
oxidation have been suggested to result in increased availability of fatty acids for lipid
accumulation. Fatty acid metabolites are thought to induce insulin resistance in both
liver and muscle by impairing the insulin-signaling pathway.9
Evidences suggest that Visceral adiposity, one of the most important cardiometabolic
risk factors originates from a chronic imbalance between energy intake and
expenditure.10
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At the cellular level, various pathophysiological events responsible for metabolic
abnormalities have been hypothesized. Among these, impaired cellular metabolism
has been suggested as a key pathophysiological process. In particular, defective
oxidative metabolism suggesting impairment in mitochondrial function. seems to be
involved in visceral fat gain and in the development of insulin resistance11
, Evidences
are emerging to support the concept that alterations in mitochondrial biogenesis and
energetics are linked to the development and progression of metabolic disorders and
CVD in obesity.12, 13
Both obesity and common forms of type-2 diabetes have been found to be associated
with reduction in mitochondrial size and number, which in turn results into reduced
activity of tricarboxylic acid cycle, E- oxidation, electron transport enzymes and
glucose oxidation. In diabetes mellitus, a potential role of deregulation of expression
of genes involved in oxidative metabolism has also been suggested by studies in
animals and human.14-17
.
It has also been hypothesized that the abnormally high availability of fat as a substrate
for energy leads to reduction in oxidative capacity of mitochondria and
hyperinsulinemia.18
This vicious cycle of lower energy expenditure leading to accumulation of fat and
further reduction of oxidative capacity of mitochondria eventually culminates into
visceral obesity, insulin resistance, hypertension, impaired lipid profile, thus overall
increased cardiometabolic risk.
There is clearly a significant unmet medical need for safe and effective weight-
reducing therapies to prevent the debilitating metabolic diseases and mortality that are
associated with increasing central adiposity.
To date, pharmacological agents for the management of Cardiometabolic risk have
been limited and unsatisfactory. Most have attempted to address weight reduction by
targeting appetite at the central nervous system.19
So far most of these with central
effect have been plagued by a higher incidence of depressive symptoms or have
resulted in higher incidence of cardiovascular side-effects.
Thyroid hormones (THs) play an important role in several physiological processes
including growth and development in early life as well as metabolic control later in
life. The mechanism underlying the calorigenic effects of THs remains to be
elucidated. Among the widespread actions of thyroid hormones (THs), increasing
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metabolic rate20
and lowering atherogenic serum lipoproteins are clinically interesting
metabolic responses since these could hypothetically be of use to treat obesity and its
related co-morbidities21, 22
. However previous attempts to mimic the effects of THs
using thyroid hormone metabolites and analogues have been complicated by the
induction of thyrotoxic adverse effects in other organ systems such as the heart and
skeleton23, 24
. Consequently, the development of a TH agonist or analogue that retains
anti obesity efficacy while being devoid of thyrotoxic effects, would represent a
potentially valuable therapeutic approach for obesity related co-morbidity. T2 (3.5-
diiodothyronine) is a metabolically active iodothyronine with the capacity to stimulate
oxidation of fatty acids in metabolically active tissues (heart, skeletal muscle, liver
and Brown adipose tissue) at relatively low concentrations. T2 has lesser affinity
towards the thyroid receptor compared to T3 and its metabolic effects are rapid 25
.
TRC150094 is a novel thyromimetic analogous to 3.5-diiodothyronine (T2) being
developed by Torrent for the treatment of Cardiometabolic risk associated with non-
traditional risk factors.
TRC150094 is a thyromimetic analogous to T2 that aims at increasing the energy
expenditure and thus regaining the balance.
In various preclinical studies conducted in multiple animal models of visceral
adiposity, insulin resistance and metabolic syndrome, it was found that TRC150094
increases energy expenditure through increase in mitochondrial metabolic activity and
attenuates visceral adiposity, atherogenic dyslipidemia, blood pressure and improved
insulin sensitivity.
If these effects are replicated in clinical setting, this profile will prove invaluable in
the treatment of Cardiometabolic risk associated with visceral adiposity.
Clinically, the continuous administration of TRC150094 administration leading to
sustained increase in Energy Expenditure is expected to result in:
• Decrease in adiposity
leading to a more desirable body mass
composition,
• Decrease in hepatic fat thus improvement in NAFLD,
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• Improvement in insulin sensitivity thus improvement in glucose
homeostasis,
• Improvement in insulin sensitivity, dependent renal-hormonal axis
(RAAS and SNS), endothelial function and thus, improvement in
Blood Pressure profile,
• Improvement in lipid profile.
Phase 1 Single Dose studies have been completed that included Single
Ascending dose study in overweight/obese subjects, food effect study and
elderly study. During these studies, TRC150094 has been found to be well
tolerated; with safety established up to 400 mg single dose in overweight/
obese subjects of age group 18-65 years. Multiple Ascending Dose study is
ongoing. There were two sequential dose levels (50 mg OD and 150 mg OD
for 28 days) planned for this study. Of these, the first cohort (50 mg OD for 28
days) has been completed and the safety has been established on this dose.
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3.1 Rationale for Current Study
Insulin resistance (IR) is a major factor responsible for Cardiometabolic risk and
consists of two important features: peripheral and hepatic insulin resistance.
Peripheral insulin resistance leads to decreased insulin stimulated glucose uptake in
skeletal muscle and adipocytes as well as decreased insulin-mediated suppression of
free fatty acids (FFA)-release. Hepatic insulin resistance increases plasma glucose
levels by decreasing insulin mediated suppression of glucose production. The
physiological mechanisms underlying insulin resistance are complex and not fully
elucidated. Amongst others, an impairment of the skeletal muscle metabolic energy
transduction pathways (mitochondrial dysfunction) has been suggested to be causally
related to insulin resistance9 Mitochondrial and cytosolic fatty acid overload and
incomplete fatty acid oxidation have been suggested to result in increased availability
of fatty acids for lipid accumulation. Fatty acid metabolites are thought to induce
insulin resistance in both liver and muscle by impairing the insulin-signaling pathway.
Since T2 has been shown to activate processes enhancing fatty acid oxidation and
thermogenesis, T2 could play a role in decreasing fat accumulation in muscle and
liver and improve insulin sensitivity. Indeed TRC150094 has been shown to improve
insulin sensitivity in preclinical studies. Whether TRC150094 will also improve
insulin resistance in clinical studies remains to be elucidated. We therefore propose to
investigate the safety and efficacy of treatment with TRC 150094 on increasing
peripheral and hepatic insulin sensitivity as well as lowering muscle and liver
triglyceride concentration in patients with the metabolic syndrome. The current Phase
2A study is designed to investigate safety and efficacy of TRC150094 for increasing
insulin sensitivity in patients with increased cardiometabolic risk. This study will be a
double-blind, randomized, placebo-controlled, multiple-dose, parallel study to assess
effect on insulin sensitivity and to explore early efficacy markers (hepatic fat, lipid
parameters and metabolic markers) after oral administration of TRC150094 tablet for
28 days. The tentative dose will be 50mg OD. This dose level has been calculated
based on the assessment of safety and PK data obtained from SAD study. The exact
dose level will be confirmed on the basis of interim safety and PK data obtained from
MAD study. Full discussion of the study design is presented in Section 5.
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4. OBJECTIVES
4.1 Primary Objective
To determine the safety and efficacy (in increasing insulin sensitivity) of TRC150094
once daily dosing for 4 weeks in male patients with increased cardiometabolic risk.
4.2 Secondary Objectives
• To evaluate the effect of TRC150094 on hepatic fat and metabolic parameters.
• To evaluate the ethnic differences for effect of TRC 150094 on Insulin
sensitivity parameters
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5. STUDY DESIGN
5.1 Overview
This will be a Phase 2A, two-centre, double-blind, placebo-controlled, multiple-dose
study to assess the effect of multiple oral doses of TRC150094 on insulin sensitivity
in 40 overweight/obese male subjects. 20 Subjects will be enrolled in India and
another 20 subjects at Amsterdam, the Netherlands. The maximum duration of
participation in the study for each subject will be 17.5 weeks including a ≤12 weeks
screening period, 4 weeks of treatment and a 10 days post treatment follow-up
evaluation period (see Appendix B for Study Assessments).
At each study site 20 subjects will be enrolled. Each subject will attend the study
centre in a fasting state, for a screening visit, 2 study visits (one baseline and one end
of treatment), 1 intermediate safety visit and 1 post-study follow-up visit (Total 5
visits). The subjects at each site will be randomized to receive TRC150094 or placebo
in a ratio of 1:1. The tentative dose level is 50 mg to be administered OD (morning)
under fasting conditions. Dosing will take place daily on Days 1−28. Subjects will
arrive at the study centre for screening visit. Physical examination, vital signs, safety
biochemistry and laboratory investigations for verification of inclusion/ exclusion
criteria will be performed during screening visit. Subjects meeting all the inclusion
criteria and none of the exclusion criteria and who have given their informed consent
for the study will be asked to come for the study on Day 0 (or day -1 if required).
Baseline investigations (including baseline clamp procedure and hepatic MRS) will be
done on Day 0 (or day -1). Subjects will receive properly labelled bottle containing
either Active treatment or Placebo as per the randomization number of the subject.
Subjects will be asked to take the dose (tentatively 50mg) OD in the morning on Days
1-28 inclusive, in fasting conditions, with a glass of water. Drug compliance of at
least 90% will be ensured. Hence the allowable limit of missing the dose should not
be more than 3 days in total. Discontinuation should not be of more than 2
consecutive days at any point of time. Uniformity in timing of intake of medication
will be advised which should be preferably within ± 1 hr of the time of intake of study
medication on Day 1. Each subject will attend the study centre on Day 14 for safety
investigations. A deviation of ± 1 day will be allowed for these visits. Subjects will
attend the study centre again on Day 28 for clamp procedure and hepatic MRS. A
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deviation up to +2 days is acceptable only in those cases that missed doses within the
limits given above and has continued planned medication up to day of post-dose
investigations. For further details, see Section 8.4 and Appendix A. It is planned that
the study will take place over 6 months (including screening and follow-up periods).
5.2 Rationale for Study Design, Control, Doses and Study Population
The tentative dose (50 mg) has been selected based on estimates of safety from pre-
clinical studies, and safety and tolerability data obtained from SAD study. The study
will begin subject to the availability of safety data of relevant dose of MAD study
(currently ongoing). A placebo control has been included in the study design to allow
for an unbiased assessment of insulin sensitivity. The study will be double-blind and
randomized to ensure unbiased data.
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6. STUDY POPULATION AND TREATMENT
6.1 Number and Description of Subjects
It is intended that 40 subjects complete the study: 20 subjects in India and 20 subjects
in Amsterdam, the Netherlands. An attempt will be made to dose 20 subjects at each
study site (10 for Active treatment; 10 for Placebo treatment). Subjects will be
identified by their initials. In addition, subjects will be given a screening number at
screening and a subject (randomisation) number at the time of randomisation. A list
identifying the subjects by initials, screening number, subject number and status
(completed study/withdrawn) will be kept in the trial master file.
6.2 Inclusion Criteria
Subjects will be considered eligible for entry in the study if they meet all of the
following criteria.
1. Adult male
2. Age range 30−65 years at screening
3. Caucasian or Indian Ethnicity
4. Waist circumference ≥ 102 cm for Caucasians and ≥ 90 cm for Indians at
screening.27
5. Fasting Serum Insulin ≥ 10 mU/ml at screening
6. Blood Pressure ≥ 130/85 mmHg at screening(or patients taking medication for
hypertension)
7. Stable weight during 3 months prior to the study (assessed through medical
history of the patient)
8. Drug naïve diabetic patients* or patients with impaired fasting glucose i.e >
100 mg/dl or 5.5 mmol/l and < 200 mg/dl or 11.0 mmol/l. Diabetic patients
who were taking metformin and have undergone washout for at least 4 weeks
before Day 0 and are currently on life style modification as a treatment for
diabetes will also be allowed in the study
9. Willingness to give written informed consent (prior to any study-related
procedures being performed) and ability to adhere to the study restrictions and
assessments schedule.
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* Diabetic patient is defined as a patient with a documented history of type II
DM or a documented history of a fasting glucose > 200 mg/dl or 11.0 mmol/l or
2x fasting glucose > 126 mg/dl or 6.9 mmol/l (2x = recorded twice)
6.3 Exclusion Criteria
Subjects will not be considered eligible for entry in the study if they meet one or more
of the following criteria.
1. Medical history, physical examination, vital signs, clinical laboratory tests, 12-
lead ECG and Chest X ray (in India only) with any significant abnormalities,
in the opinion of the investigator.
2. Subjects with any known somatic illness, including neoplasm, endocrine
disorder such as cushing’s disease, PCOD and uncontrolled hypothyroidism,
neurologic disorder, active infection, or recent surgical procedure within 3
months of the study initiation.
3. Subject currently using medication, which can influence glucose or FFA
metabolism such as fibrates, niacin, ACE inhibitors (subjects are not excluded
in case of use of a stable dose for at least 6 weeks prior to baseline
measurement, taking (ACE) inhibitors (ACE-I) or angiotensin-receptor
blockers (ARBs)), PPAR agonists, omega 3 fatty acids.
4. eGFR < 60 mL/min/1.73m2 at screening as evaluated by Modification of Diet
in Renal Disease (MDRD) method.
5. History of angina, Myocardial Infarction (MI) or stroke since last 6 months.
6. Hypertension with SBP/DBP ≥160/100 mm Hg at screening.
7. ALT or AST ≥ ULN*3 at screening
8. History or presence of malignancy.
9. History of recreational drug use within the last 30 days, or regular
consumption of greater than 2 units of alcohol/day
10. History of allergy to the test drug or any drug chemically similar to the drug
under investigation.
11. Seropositive for Hepatitis B, Hepatitis C or HIV.
12. Subjects suffering from any psychiatric (acute or chronic) illness.
13. Intake of any medication except those permitted in this study (see Section 6.6).
14. Intake of any investigational drug in the period within 3 months prior to the
first dose of study drug.
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15. History of significant blood loss due to any reason, including blood donation,
in the 12 weeks prior to the first dose of study drug; or the total blood loss in
the last 3 months, including for this study, exceeds 450 mL.
16. History of any bleeding disorder.
17. Existence of any surgical or medical condition which, in the judgment of the
principal investigator, might interfere with the absorption, distribution,
metabolism or excretion of the study drug or might be likely to compromise
the safety of the subject.
18. Inability to communicate or co-operate with the investigator because of
language problems, poor mental development or impaired cerebral function.
19. Inability to comply with study requirements.
20. Positive drugs of abuse test (at screening) and alcohol breath test.
21. Heavy smokers (who are smoking >15 cigarettes or equivalent per day).
6.4 Sample Size Calculation
It is intended that 40 subjects (20 subjects in India and other 20 in Amsterdam, the
Netherlands) will complete the study. No formal sample size calculation has been
performed. We calculated that a sample size of 20 in each group will have 80% power
to detect an absolute difference in Rd of at least 15 µmol/kg·min (measure for
peripheral insulin sensitivity), before and after treatment, using a Wilcoxon (Mann-
Whitney) rank-sum test with a 0.05 two-sided significance level and assuming that the
common standard deviation is 15.
6.5 Investigational product
Subjects will be treated with either TRC150094, or a placebo for 28 days.
TRC150094 is a novel synthetic compound, chemically, 3-[4-(7-Hydroxy-6-methyl-
indan-4-ylmethyl)-3,5-dimethyl-pyrazol-1-yl]-propionic acid, which is currently in
clinical development for the treatment of Increased Cardiometabolic risk associated
with non-traditional risk factors.
6.6 Prior and Concomitant Medication
Subjects are not permitted to enter the study if they have taken any investigational
drug in the 3 months prior to study drug administration. Subjects are not permitted to
enter the study if they have taken insulin or any oral anti-diabetic (except metformin)
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in past. Those subjects who are taking metformin may be included in the study after a
washout of at least 4 weeks. During these 4 weeks, these subjects should be on
lifestyle modification as a treatment for diabetes. Other concomitant medications may
be allowed after agreement between principal investigator and sponsor. Prior
medications (up to 4 weeks prior to the dosing occasion) and all concomitant
medications (up to the post-study follow-up visit) will be recorded in the subject’s
case report form (CRF).
6.7 Restrictions on Subjects
6.7.1 Diet
All subjects will fast for at least 10 hrs before Screening. Subjects will fast for
approximately 13 hrs before Day 0 and Day 28 investigations. During the dosing
period, the subjects will be instructed to fast for at least 1 hr prior to dosing (morning)
and for 1 hr post-dose. Subjects are required to present to the research centre after an
overnight fast of at least 10 hrs for Day 14±1 and follow-up visits.
6.7.2 Alcohol, caffeine, grapefruit
Subjects are not permitted any alcohol or caffeine-containing food or drinks from 48
hrs prior to study investigation (Day 0) until discharge before dosing period and
similarly, from 48 hrs prior to post dose investigation (Day 28) until discharge. In
addition, no alcohol is permitted for 48 hrs before the screening, Day 14±1 and
follow-up visits. While resident in the CRU, all drinks and food will be decaffeinated.
Subjects are permitted up to 6 caffeinated drinks per day in the period from screening
to 48 hrs prior to admission for baseline investigation, during Day 1 to Day 26 and
from discharge to the follow-up visit. Subjects are not permitted any grapefruit or
grapefruit-containing food or drinks from 1 week prior to dosing (first study drug
administration) until the post-study follow-up visit.
6.7.3 Exercise
Subjects must refrain from strenuous exercise for 48 hrs prior to the screening visit.
Subjects must not undertake strenuous and/or unaccustomed exercise from 1 week
prior to study investigation (Day 0). They are however allowed to undertake mild and
accustomed exercise during their period of dosing (Day 1 to Day 28). Subjects must
also refrain from strenuous and/or unaccustomed exercise during the period between
discharge and follow-up visit.
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7. INVESTIGATIONAL PRODUCT ADMINISTRATION
7.1 Description of investigational products
Active study drug:
Laboratory Code: TRC150094.
Excipients: Cellulose Microcrystalline, Hypromellose, Magnesium stearate
Formulation: Tablet
Strengths: 2.5 mg, 12.5 mg, 50 mg
Route of administration: Oral
Placebo tablets:
Contents: Cellulose Microcrystalline, Magnesium stearate
Formulation: Tablet
Route of administration: Oral
Placebo tablet will be administered orally (tablet will have an identical
appearance and volume to TRC150094 tablet).
7.2 Summary of pharmaceutical, nonclinical and clinical information of
TRC150094
7.2.1 Physical, Chemical & Pharmaceutical summary
TRC150094 is white to off-white powder, soluble in DMSO and practically insoluble
in water. TRC150094 Tablets are white to off-white, round, uncoated tablets.
TRC150094 tablets (all strengths) should be stored at room temperature not exceeding
25°C and protected from direct sunlight.
7.2.2 Nonclinical Studies
Pharmacology
To assess the potential of TRC150094 for the treatment of adiposity associated
metabolic disorder, it was administered to diet induced obese C57BL6 mice (DIO
mice), which simulate the pathophysiological features of human metabolic disorder.
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The study revealed dose dependent increase in oxygen consumption and significant
attenuation of body weight gain in TRC150094 treated group. Mice treated with
TRC150094 had significantly (p<0.05) less abdominal, epididymal, perirenal,
scapular, lumbar and total fat pad in comparison to control. TRC150094 treated group
also showed significantly reduced LDL-C as compared to control. Fasting plasma
insulin and glucose levels were estimated and HOMA-IR index was derived. HOMA-
IR index was found to be lower in TRC150094 treated group as compared to control,
thus indicating improved insulin sensitivity. TRC150094 has also shown to
significantly reduce hepatic fat content as compared to control. Thus TRC150094 has
shown beneficial effect on the individual components of metabolic syndrome that is
adiposity, insulin sensitivity, lipid profile and hepatic fat in DIO mice. In another 4
week TRC150094 study on wistar rats fed on High fat diet, it was found that the
treatment group had significantly less visceral adipose tissue than control. In liver,
mitochondrial fatty acid import and oxidation were increased as compared to control,
and consequently the hepatic triglyceride content was lower. These effects were
independent of the AMP-activated protein kinase-acetyl CoA-carboxylase-malonyl
CoA pathway but involved sirtuin-1 activation. In skeletal muscle, TRC induced a
fiber shift toward the oxidative type in tibialis anterior muscle, increasing its capacity
to oxidize fatty acids. HFD-TRC rats had lower (vs. HFD rats) plasma cholesterol and
triglyceride concentrations.26
Long term in vivo efficacy studies of TRC150094 were
conducted using different animal models such as DIO mice, hamster and Ob ZSF1 rat.
In the long term treatment studies, TRC150094 not only attenuated the visceral fat
accumulation but also attenuated various cardiovascular risk components associated
with visceral adiposity and type 2 diabetes. For instance, long term treatment of obese
ZSF1 rat with TRC150094 improved glucose tolerance, glycemic and lipid profile,
attenuated rise in blood pressure and improved functional capacity of skeletal muscle
without influencing appetite, thyroid hormone, cardiac function and skeleton.
Selectivity and safety pharmacology studies suggest that TRC150094 does not have
significant enzyme inhibition or ligand binding activity towards a variety of enzymes
and receptors. TRC150094 demonstrated no significant safety concerns in nonclinical
cardiovascular, respiratory and CNS safety pharmacology studies and demonstrated a
wide safety margin.
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Pharmacokinetics and Metabolism
The nonclinical pharmacokinetic and metabolic profile of TRC150094 has been
characterized. When administered orally, TRC150094 is rapidly absorbed in male
Wistar rat with an absolute bioavailability of 92%. In toxicokinetic studies the Cmax
achieved at 1-4 hrs in beagle dogs. TRC150094 has shown dose related increase in
exposure at several fold higher pharmacological doses across the species and genders.
The parent compound showed rapid and wide distribution. TRC150094 is
metabolically stable in microsomes and hepatocytes of various species tested. There is
no inhibition and induction of the major CYP450 enzymes at pharmacologically
relevant concentrations. In vivo studies in rat revealed three Phase 2 metabolites in the
form of hydroxylate, sulphate and glucuronate conjugates of the drug. TRC150094
has shown dose proportionality up to several folds of pharmacological doses across
the species and genders, without any accumulation potential. The terminal half life of
TRC150094 is 4-8 hrs in rats and 7-10 hrs in dogs. About 98% of the compound is
excreted in 48 hrs through urine and faeces in rats.
Toxicology
TRC150094 was found to be non-mutagenic in AMES test, Mouse lymphoma assay
and Micronucleus test. TRC150094 was found to be well tolerated in acute studies in
rats and mice by clinical and parenteral route. Chronic repeat dose toxicity studies
were conducted in Wistar rats (26 week) and Beagle dogs (39 week). There were no
test item related clinical signs observed in rat and dog treated chronically. However,
Beagle dogs treated in 4 week study showed emesis at 2-4 hr post dosing at high dose
of 450 mg/kg/day. Transient reduction in thyroid hormones (total and free T4) was
observed in rat studies at top doses of 180 and 360 mg/kg/day, while in dog it was
seen in 4 week study at top dose of 450 mg/kg/day. Thyroid hormone profile
remained unaltered in 39 week dog toxicity study up to a high dose of 75 mg/kg/day.
The change in thyroid hormone profile is considered as test item related
pharmacological effect. Repeated exposure of TRC150094 revealed kidney as target
organ for toxicity in both rat and dog species. Increased kidney weight was evident at
highest doses up to the chronic duration in rats. Microscopically, dilated renal tubules
and nephropathy was seen at highest doses of 180 and 360 mg/kg/day in rats. Beagle
dogs treated in 4 week study at 450 mg/kg/day revealed dilated renal tubules,
lymphocytic infiltration and nephropathy in kidney and degeneration of seminiferous
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tubules of testes. There were no such alterations observed in dog treated at high dose
of 75 mg/kg/day in 13 and 39 week studies. No observed adverse effect level
(NOAEL) doses in Wistar rat and Beagle dog is 60 and 75 mg/kg/day, respectively in
chronic toxicity studies which demonstrated very high safety margins. TRC150094
did not have any adverse impact on male and female fertility indices studied in rats
and found to be non-teratogenic in rat and rabbits.
7.2.3 Available Clinical Data
The clinical evaluation of TRC150094 to date consists of Single Dose studies and
Cohort 1 of Multiple Ascending Dose Study.
Single Dose Studies:
The Single Dose Studies included:
PART A: Single ascending dose in non-elderly and elderly overweight/obese male
and female subjects to evaluate safety, tolerability and pharmacokinetics of
TRC150094.
PART B: To evaluate effect of food on pharmacokinetics of TRC150094 in young
healthy male and female subjects and to assess the safety and tolerability of single
oral doses of TRC150094 administered in fasted and fed states.
A total of 40 subjects were randomized in Part A. Out of 40 subjects, 24 were non-
elderly overweight/obese subjects aged 18-65 years and 16 were elderly aged above
65 years. 24 subjects were divided in 3 cohorts (Cohort 1: 5 and 100mg; Cohort 2: 25
and 200 mg; Cohort 3: 50 and 400 mg) each having 8 subjects and 16 elderly subjects
were assigned to fourth cohort (50 and 150 mg). Out of 24 non-elderly subjects, 18
were administered TRC150094 and 6 were administered placebo. Among elderly
subjects, 12 were administered TRC150094 and 4 were administered placebo. Each
subject received two dose levels with a washout period of 7 days in between the two
study periods. In Part B of the study 6 healthy subjects aged 18-65 years were
randomized to receive the active dose of 100 mg in fasting and fed conditions, with a
washout period of atleast 7 days between the two study periods.
The main findings of these studies are as follows:
Safety and tolerability- The ascending doses of TRC150094 ranging from 5 mg to 400
mg, evaluated in the study were found to be safe and well tolerated in both elderly, as
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well as non-elderly overweight/obese subjects. There were no SAEs or withdrawals
due to AE in the study. All the adverse events were mild to moderate in nature. In
non-elderly cohort, headache was the most common AE experienced by 3 (8.3%)
subjects followed by decreased appetite, pain in extremity and dizziness postural,
each experienced by 2 (5.6%) subjects in the TRC150094 group. Headache was the
most common AE experienced by 2 (8.3%) subjects in the elderly cohort, also,
followed by back pain experienced by 1 (4.2%) subject receiving TRC150094. No AE
was reported in the placebo group in elderly cohort.
Pharmacokinetics- Absorption of TRC150094 was rapid (Tmax reached within 3 hrs)
and elimination half life ranged from 15-18 hrs. AUC0-∞ was found to be dose
proportional and Cmax seemed greater than dose proportional across the dose range.
More than 80% of total excreted amount (during 48 hrs) in urine was recovered by 24
hrs in non-elderly and elderly subjects. Total exposure was found to be higher in
elderly subjects compared to non-elderly subjects. Renal clearance has been observed
to be decreased in elderly subjects as compared to non-elderly subjects. Gender
related differences with Cmax and AUC0-∞ were not found in elderly and non-elderly.
Presence of food reduces Cmax by approximately 50% without any effect on total
exposure in both the genders. All metabolites found in humans have also been found
in toxicology species. Human specific metabolite was not observed. This indicates
that the animals in the toxicological studies have been exposed to the same
metabolites likely to form in clinical setting.
Multiple Ascending Dose Study-
The first cohort of this study had been completed. The first cohort comprised of 16
overweight/obese subjects, out of which there were 11 males and 05 females. Of the
16 subjects dosed, 12 subjects received active treatment and 4 subjects received
placebo. Dosing (50mg once daily) took place for 28 days (Days 1−28).
Safety and tolerability parameters (adverse event [AE], electrocardiogram [ECG],
vital signs and clinical laboratory test results) data were evaluated at weekly intervals
during the study. The safety and tolerability evaluation was done up to 48 hours post
dose (relative to the dose administered on Day 28) and of follow-up visits from the
first dose level. All these data were reviewed at regular intervals by an independent
Data Safety Monitoring Board (DSMB). All the subjects attended for a follow-up visit
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8-10 days after the last dose. There has been no safety concern and DSMB has
recommended continuation with dosing of subsequent dose level (150mg per day).
No serious adverse events (SAE) and no important medical events occurred during the
conduct of the study.
No withdrawal due to adverse events (AE) occurred during the conduct of the study.
One adverse event was observed in one subject. The details are as follows:
Randomisation number 1002
Description Cough
Start date and time 14/12/10, 2000
End date and time 16/12/10, 1130
Onset after dosing 09 hrs and 58 minutes
Duration 40 hrs and 30 minutes
Severity Mild
Relationship to study drug Unrelated
Treatment Grilinctus cough syrup
Apart from recording vitals at predefined time-points, Ambulatory Blood Pressure
and Heart rate was recorded at Baseline, Day 14 and at end of study. No clinically
relevant changes in Blood Pressure and Heart rate were observed during the study.
In addition, the effect of TRC150094 on Blood Pressure and Heart rate parameters
during exercise was evaluated at baseline and at end of study by Cardiopulmonary
Exercise testing. No clinically relevant changes have been observed.
12-lead ECG was recorded in triplicate at each time-point, with each ECG separated
by approximately 1 minute at each time point. No clinically relevant changes were
observed in ECG during the study and post study follow up. No clinically relevant
changes were observed in ECG time intervals : in particular, there was no
prolongation of PR interval > 220 msec associated with a greater than 20 msec
increase from baseline, prolongation of QRS interval > 120 msec , and of
prolongation of QTc (Bazett) interval > 450 msec in subjects.
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Cardiac monitoring by ECG was done during exercise while conducting Cardio-
pulmonary exercise test. Special attention was given to rhythm of ECG to identify any
provocable arrhythmia in these subjects. There has not been any evidence of
provocable arrhythmia or ectopics in any of the subjects dosed in first cohort.
The laboratory safety parameters in this study included renal safety markers, bone
injury markers, cardiac safety markers and ACTH apart from routine battery of safety
tests. No clinically relevant changes in laboratory tests including liver enzymes, renal
safety markers, bone injury markers, cardiac safety markers and ACTH, related to
intake of TRC150094 were observed throughout the study.
Overall, the safety and tolerability of 50 mg Dose in Multiple Ascending Dose study
has been established.
7.2.4 Summary of potential risks and benefits
The ascending doses of TRC150094 ranging from 5 mg to 400 mg, evaluated in the
SAD study were found to be safe and tolerable in non-elderly overweight/obese
subjects. TRC150094 was also found to be safe and tolerable in elderly cohort. There
were no SAEs or withdrawals due to AE in the study. All the adverse events were
mild to moderate in nature. Headache was the most common AE observed in both
elderly and non-elderly cohorts. Overall, the safety and tolerability up to 400 mg
single dose has been established. Based on this data, we have selected 50 mg multiple
dose administration (OD for 28 days) as a tentative dose level for this study which is 8
times lower than the dose at which safety in single ascending dose study has been
established. Moroever, the Cohort 1 of MAD study has been completed and the safety
and tolerability of multiple administrations of 50 mg OD dose level has been
established.
7.2.5 Description and justification of route of administration and dosage
The planned dose has been selected based on estimates of safety from pre-clinical
studies and safety and tolerability data obtained from SAD study. The study will
begin subject to the availability of safety data of 50 mg dose of MAD study. A
placebo control has been included in the study design to allow for an unbiased
assessment of insulin sensitivity. This part of the study will also be double-blind and
randomized to ensure unbiased data.
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7.3 Dose Administration
Dosing will start on Day 1. It is planned that each subject will receive once daily
dosing (tentatively 50 mg) in the morning for Day 1-28 under fasting conditions with
a glass of water. Uniformity in timing of intake of medication will be advised which
should be preferably within ± 1 hr of the time of intake of study medication on Day 1.
The dosing will not be under direct supervision (except Day 28 dosing); the subjects
are required to maintain a diary where they will make entry for the daily dosing. The
subjects will be advised not to take any food for at least 1 hr prior to dosing and for 1
hr post-dose. For outpatient days (i.e. on days 1 to 28) drug supply in appropriately
labelled bottles will be provided to the subjects. The subjects will be instructed to
carefully document the time of drug consumed and time of meals and exercise, in the
diary cards provided to them. Subjects will be asked to bring the remaining drugs with
the diary cards during intermediary visit to the clinic. The study pharmacist or
technician will carry out drug reconciliation and ensure compliance of the subject is
satisfactory. Appropriate documentation of the subject specific dispensing process
must be maintained. The batch number of each study drug administered to each
subject will be recorded in Pharmacy documents.
7.4 Supply, Identification and Storage
The investigational products will be manufactured, handled and stored in accordance
with Good Manufacturing Practice (GMP) and used in accordance with this protocol.
Torrent Pharmaceuticals Limited (sponsor) will manufacture and provide the
TRC150094 and placebo tablets and will ensure that the drug supplies are suitable for
human use.
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The sponsor will supply Certificates of Analysis for the TRC150094 and placebo
tablets, including the batch numbers and expiry dates. The bulk supply will include
TRC150094 tablets and Placebo tablets. The labels of the bulk packaging supplied by
the sponsor will include the following information:
• Name, address and telephone number of sponsor
• Trial reference code
• Investigator name
• Name of study drug and formulation
• Dose strength
• Route of administration
• Batch number
• Manufacture date
• Expiry date/re-test period
• Storage and reconstitution instructions
• “For Clinical Trial Use only”
• “Keep all medicines out of reach of children”
On receipt, the pharmacy staff at the CRU will check the contents and will complete
the appropriate documentation. The study drugs will be kept in a secure, temperature-
controlled, restricted-access location and in accordance with applicable regulatory
requirements. The study drugs will be stored at a temperature below 25°C, and
temperature logs will be maintained. The investigator will ensure that the
investigational products are used only in accordance with this protocol.
7.5 Treatment Compliance
Details of study drug administration will be recorded in subject diary. The same will
be transcribed to CRF. History of drug compliance will be recorded during the interim
safety visit at Day 14±1 and Day 28+2. Drug compliance of at least 90% will be
ensured. Hence the allowable limit of missing the dose should not be more than 3
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days in total. Discontinuation should not be of more than 2 consecutive days at any
point of time.
7.6 Treatment of Overdose
In the event of an overdose of study drug, the subject should be given supportive
treatment depending on the symptoms.
7.7 Accountability
The pharmacy staff and the investigator at the CRU will be responsible for drug
accountability. Records will be kept of:
• all study drugs delivered to the trial site
• all study drugs dispensed (with batch no.)
• the administration to each subject
• all study drugs remaining and returned/destroyed.
These records will include dates, quantities, batch numbers, expiry dates, and the
unique code numbers assigned to the investigational products and trial subjects.
Monitor assigned by sponsor (who is not directly associated with conduct of study)
will verify the drug accountability records during the study and will perform drug
reconciliation at the end of the study. At the end of the study, unused study drugs will
be returned to the sponsor or destroyed, as directed by the sponsor (by written
authorisation). The return/destruction of all investigational medicinal products will be
documented appropriately.
7.8 Dispensing of Investigational Product
The dispensing pharmacist will dispense a quantity of the investigational products
sufficient for dose administration well in advance as per the randomization schedule
in presence of Quality Assurance (QA) and the remaining Investigational Products
will be kept in their original containers. Individual subject supplies for each visit in
HDPE bottles will be packed and labelled according to local regulations. For
dispensing at Veeda, each subject bottle will contain sixteen tablets (14 for study drug
medication once daily and 2 for extra) per visit while for dispensing at AMC, each
subject will be provided bottles with 30 tablets/bottle (including 2 reserve tablets). For
last dosing on day 28, subject will visit the site on day 27 and subject will be asked to
take a tablet (last dose) under supervision.
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8. METHODS
8.1 Study parameters/endpoints
8.1.1 Main study parameter/endpoint
To determine safety and efficacy for TRC150094 in increasing hepatic and peripheral
insulin sensitivity
8.1.2 Secondary study parameters/endpoints
To determine the effect of TRC150094 on hepatic fat and multiple metabolic
parameters
8.2 Randomisation and treatment allocation
8.2.1 Preparation of randomisation code
The randomisation code and sealed individual code-break envelopes will be prepared
by the clinical research unit at each site. One set of the individual code-break
envelopes will be prepared and provided to the investigator. Each research unit will
prepare three randomization lists for statistician, pharmacist and sponsor respectively.
The randomisation code will specify whether the subject receives TRC150094 or
placebo dosing over the course of the study period. The randomisation code will be in
consecutive sequence at the two study centres; hence at one unit it will be 001-020
and other unit it will be 021-040. The randomization list provided to the sponsor
nominated individual (who is not directly associated with the study) will be sealed.
The seal will be ensured at the end of study. All randomisation information will be
secured and kept in a locked storage area, accessible only by authorised personnel.
Only subjects who meet all of the inclusion criteria and none of the exclusion criteria
are eligible for randomisation. Randomisation will take place on Day 0, the day
before dosing. Subjects will be randomized to receive either placebo or TRC 150094
in 1:1 ratio. Each subject will be given a subject number (equivalent to the
randomisation number). This number will be a consecutive number from 001
onwards. It will be assigned in the order of the inclusion of subjects at the first dosing.
8.2.2 Breaking the randomisation code
The investigator will have access to one set of sealed code-break envelopes. In the
event of an emergency, e.g. a serious adverse event (SAE), the investigator may open
the subject’s envelope to determine the study drug administered to the subject. If
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possible, the sponsor should be notified before the blind is broken; otherwise, the
sponsor will be notified as soon as possible. In addition the IEC (Independent Ethics
Committee) will also be notified if the code is broken during the study. The date, time
and reason, and the name of the person who opened the envelope, will be recorded in
the subject’s CRF and on the individual envelope. If the blind is broken, the subject
must be withdrawn from the study and replaced.
8.3 Blinding
This will be a double-blind design. The subjects and investigator (and other personnel
involved in the study) will be unaware of the study drugs administered to individual
subjects.
Personnel in the biometrics department at Academic Medical Centre and Veeda
Clinical Research will be blind during the study, except for a nominated individual(s)
responsible for preparing the randomisation code and code-break envelopes. The
sponsor will remain blind during the study. The placebo tablets will be identical in
appearance and taste to the TRC150094 tablets administered at the corresponding
dose level, and the same number will be administered. Pharmacy personnel at the
study centre who prepare and check the individual unit doses from the bulk packaging
will be the only staff at the study centre that is not blind to the study drug. At regular
monitoring visits, a study monitor will check the blind is maintained during dosing
and during the study, and that all code-break envelopes remain intact. The monitor
will remain blind during the study, and steps must be taken during monitoring such
that treatment allocation is not revealed.
8.4 Study Procedures
This section describes the Hyperinsulinemic Euglycemic Clamp and Magnetic
resonance spectroscopy (MRS) procedures
8.4.1 Hyperinsulinemic Euglycemic Clamp
The clamp procedure will be performed twice on each subject (on Day 0 and Day 28)
after an overnight fast. A deviation up to +2 days is acceptable only in those cases that
missed doses within the limits given above and has continued planned medication up
to day of post-dose investigations. Participants will be admitted to the trial unit and
studied in the supine position. During the procedure, the participants are only allowed
to drink water. Following a 13 hr fast, a catheter will be inserted in the vein of each
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arm. One catheter is used for sampling of arterialised blood using a heated hand box
(60°C). The other catheter is used for infusion of [6,6-2H2]-glucose en and [1,1,2,3,3-
2H2]-glycerol, glucose 20% and insulin. At t= -2hr, after drawing a blood sample for
background enrichment of plasma glucose, a continuous infusion of [6,6-2H2]-
glucose (>99% enriched, Cambridge Isotopes, Massachusetts, USA) is started at a
rate of 0.11 µmol/kg per min after a priming dose equivalent to 100 min of infusion.
After 1 hr, a continuous infusion of [1,1,2,3,3-2H2]-glycerol (>99% ernriched,
Cambridge Isotopes, Massachusetts, USA) is started at a rate of 0.11 µmol/kg per min
after a priming dose of 1.6 µmol/kg/min. The first 2 infusion hrs are required for
equilibration. After 115, 120 and 125 min blood samples are drawn for determination
of glucose and glycerol enrichment to calculate basal endogenous glucose production
and lipolysis, glucoregulatory hormones and FFA10
. Subsequently, the
hyperinsulinemic clamp will start with a continuous infusion of insulin (Actrapid
100U/ml, Novo Nordisk ) for 2hr at a rate of 20mU/m2 body surface area per min.
Plasma glucose is measured every 10 min (Biosen C-line plus glucose analyzer; EKF
Diagnostics, Barleben/Magdeburg, Germany at AMC, Netherlands; and YSI analyzer
at Veeda Clinical Research India) and glucose 20% is infused at a variable rate to
maintain plasma glucose at 5.0 mmol/l or 90 mg/dl. [6,6-2H2]-glucose is added to the
20% glucose solution to achieve glucose enrichments of 1% to minimize changes in
isotopic enrichment due to changes in the infusion rate of exogenous glucose, and
thus to allow for accurate quantification of endogenous glucose production and
uptake10
. During the last 20 min of the hyperinsulinemic clamp, blood samples are
drawn at 5 min intervals for determination of glucose and glycerol enrichment,
glucoregulatory hormones and FFA. At t= 2.15hr, the infusion of insulin is increased
to a rate of 60mU/m2 body surface area per min. Plasma glucose is measured again
every 10 min and enriched glucose 20% is infused at a variable rate to maintain
plasma glucose at 5.0 mmol/l or 90 mg/dl. During the last 20 min of this insulin
infusion step, blood samples are drawn at 5 min intervals for determination of glucose
and glycerol enrichment, glucoregulatory hormones and FFA. Thereafter insulin
infusion will be discontinued and participants will be offered a carbohydrate rich
meal. The glucose infusion rate will be doubled. Plasma glucose levels will be
measured every 10 minutes for the first hour. Thereafter blood glucose measurement
will be done at 20 minute intervals or more frequently (at the discretion of the
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investigator). Every time plasma glucose levels are > 7 mmol/l the glucose infusion
rate will be halved until less than 5 ml/hr. Four weeks later, the second clamp will be
performed with the same methodology.
Name and description of investigational product(s)
• [6,6-2H2] labelled glucose, Cambridge isotopes, Cambridge, Massachusetts,
USA
• [1,1,2,3,3-2H2] labelled glycerol, Cambridge isotopes, Cambridge,
Massachusetts, USA
• Actrapid, Novo Nordisk
• Glucose 20%
[6,6-2H2] labelled glucose and [1,1,2,3,3-2H2] labelled glycerol will be involved at
the pharmacy at the study centre and will be prepared by the investigators.
Endogenous glucose production (EGP) and peripheral glucose uptake (Rd) are
calculated using the modified form of the Steele equations.28
Lipolysis (glycerol
turnover) will be calculated by using formulas for steady state kinetics adapted for
stable isotopes.
8.4.2 Quantification of Hepatic Fat.
Hepatic will be measured by 1H MRS on Day 0 and Day 28. A deviation ± 2 days is
acceptable. The analysis and interpretation of MRS data will be done by core lab of
Academic Medical Centre at Amsterdam, The Netherlands. During the measurements,
the subjects will lie down inside the
magnet of a clinical magnetic resonance
spectrometer with a body array surface coil positioned around the abdomen. For the
quantification of liver fat, localized single voxel proton MR spectra from the liver will
be obtained without water suppression using long repetition time and short echo time
to minimize relaxation effects on signal intensity. The MRS measurement will be
carried out without breath holding Spectral signal intensities of water and methylene
groups originating from the lipids will be fitted to determine the percentage of
intracellular triglycerides of the hepatocytes14
.
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8.4.3 Silent Information Regulator T (SIRT) expression study:
Modulation of SIRT expression will be monitored for Indian cohorts only. For this
purpose, 10ml of heparinised blood will be collected. Peripheral blood mononuclear
cells (PBMNCs) will be isolated using HISTOPAQUE within 2 hrs of blood
collection. Isolated PBMNCs will be stored at -80ºC. Total RNA will be isolated from
PBMNCs and SIRT gene expression will be monitored employing specific probes by
real-time PCR. Modulation of SIRT expression will be monitored for each volunteer
at Day 0, Day 14 and Day 28 of the study. A deviation of ±1 day is acceptable.
Analysis of samples for SIRT expression will be performed at Cell and Molecular
Biology Lab, Torrent, India.
Total 30 ml heparinised blood per volunteer will be required.
8.4.4 Measurement of Sagittal Abdominal diameter
Sagittal abdominal diameter will be measured in the supine position with bent knees
on a firm examination table. The measurement will be done to the nearest 0.1 cm after
a normal expiration and without clothes in the measurement area. Sagittal abdominal
diameter will be measured at the level of iliac crest (L4–5 using a sliding-beam
caliper, as the distance between the examination table up to the horizontal level,
allowing the caliper arm to touch the abdomen slightly but without compression29
.
8.5 Study Visits
Screening
Subjects will attend the CRU for a screening visit up to 28 days before their Baseline
investigation (Day 0). Prior to attending for a screening visit, subjects will be asked to
comply with certain exercise and dietary restrictions, as specified in Section 6.7.
Informed consent must be obtained at this visit before any study procedures are
performed. Further details regarding informed consent are provided in Section 11.2. A
screening log will be kept to record subjects who sign the informed consent form and
who are screened. For those subjects who are screen failures, a reason will be
documented. The following assessments/information will be performed/ recorded
• date of birth and age
• race
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• height and body weight (measured while wearing indoor clothing and no
shoes)
• waist circumference
• body mass index (BMI), calculated as weight (kg)/height (m)2
• smoking status / intake of tobacco in any other form (including current and
historical use of tobacco, and ability to stop smoking / tobacco intake in any
other form, for study periods)
• medical history (any significant conditions or diseases that stopped at or prior
to screening)
• pre-existing (concurrent) conditions (those present at the screening visit)
• prior and ongoing medication (medications taken up to 4 weeks prior to first
dose)
• full physical examination
• vital signs (supine and standing) and body temperature
• 12-lead ECG
• chest X-ray (to exclude active tuberculosis, in India only)
• clinical laboratory tests (blood sample for haematology and biochemistry tests;
and urine sample for urinalysis)
• lipid Parameters
• viral serology (hepatitis B surface antigen [HBsAg], hepatitis C antibody,
human immunodeficiency virus [HIV I and HIV II])
• alcohol breath test
• urinary drugs of abuse screen (amphetamines, barbiturates, benzodiazepines,
cannabinoids, cocaine, opiates and methadone).
Those subjects who are eligible for the study, based on the inclusion and exclusion
criteria, will be invited to take part in the study. Lifestyle instructions as mentioned in
Section 6.7 will be given.
Day 0
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Those subjects who meet all inclusion and none of the exclusion criteria and who
have given their informed consent will be asked to come to the CRU for baseline
investigations on Day 0. On Day 0 subjects will visit the study centre early in the
morning after a 13 hr fast. If it is not possible for a subject to arrive at the study centre
this early in the morning, the subject is offered to stay overnight. The subjects may be
asked to visit the CRU on Day-1 for some baseline investigations (at the discretion of
Principal Investigator). Randomisation will take place on Day 0.
At Day 0 the following assessments will be performed
• physical examination
• vital signs
• 12-lead ECG
• height and body weight
• waist circumference
• Sagittal abdominal diameter
• clinical laboratory tests (Haematology, Biochemistry, urine analysis)
• Blood Sample for SIRT expression (see Appendix B)
• lipid Parameters
• hepatic MRS – See Section 8.4.2 (a deviation of ± 2 days is acceptable for
MRS
• compliance to drug and life style instructions
• AEs and change in concomitant medications will be reported
• hyperinsulinemic euglycemic clamp, the clamp procedure is described in
Section 8.4.1
Treatment Period Day 1-28
Each subject will receive once daily dosing in the morning for Day 1-28 under fasting
conditions (see Section 7.5)
Day 14
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On Day 14, subjects will visit the CRU after an overnight fast. A deviation ± 1 day is
acceptable. The following assessments will be performed:
• vital signs
• height and body weight
• waist circumference
• Safety clinical laboratory tests (Haematology, Biochemistry, urine analysis)
• Blood Sample for SIRT expression (see Appendix B)
• lipid Parameters
• 12-lead ECG
• AE check and change in concomitant medications will be recorded
• compliance to drug and Life style instructions
Day 28
On Day 28 subjects will visit the study centre early in the morning after a 13 hr fast.
A deviation up to +2 days is acceptable only in those cases that missed doses within
the limits given above and has continued planned medication up to day of post-dose
investigations. If it is not possible for a subject to arrive at the study centre this early
in the morning, the subject is offered to stay overnight. The subjects may be asked to
visit the CRU on Day 27 for some end-of-treatment investigations (at the discretion of
Principal Investigator).
At Day 28 the following assessments will be performed.
• physical examination
• vital signs
• 12-lead ECG
• height and body weight
• waist circumference
• Sagittal abdominal diameter
• clinical laboratory tests (Haematology, Biochemistry, urine analysis)
• Blood Sample for SIRT expression (see Appendix B)
• lipid Parameters
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• hepatic MRS– See Section 8.4.2 (a deviation of ± 2 days is acceptable for
MRS)
• AEs and change in concomitant medications will be reported
• hyperinsulinemic euglycemic clamp, the clamp procedure is described in
Section 8.4.1
• Compliance to drug and Life style instructions
• Discontinuation of study drug
At follow up visit (Day 35), subjects will visit the study centre after an overnight fast.
Follow up visit
Follow-up visit is planned at Day 35. A deviation of + 3 days is acceptable.
At Day 35 the following assessments will be performed:
• Physical examination
• Vital signs
• Height and body weight
• Waist circumference
• Safety clinical laboratory tests (Haematology, Biochemistry, urinanalysis)
• Lipid Parameters
• 12-lead ECG
• AEs and change in concomitant medications will be reported
• Restart of metformin treatment (if applicable)
8.5.1 . Clinical laboratory tests
A blood sample will be taken for measurement of haematology and biochemistry
parameters, and a urine sample will be taken for urinalysis at the following time
points:
• screening visit
• Day 0
• Day 14
• Day 28
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• Day 35
The Laboratory parameters and time-points are specified in (Appendix A) and
(Appendix B).
8.5.2 Vital signs
Body temperature and supine vital signs (blood pressure and heart rate) will be
measured on the non-dominant arm after 5 minutes of supine rest on the Day of
screening visit, Day 0, Day 14±1 and Day 28 (t = -2hr) and at follow up (See
Appendix B). Standing vital signs (blood pressure and heart rate) will also be
measured at the screening visit. Standing vital signs will be measured after the supine
values have been recorded and after the subject has been standing for 2 minutes.
These measurements will be used to calculate the postural drop in blood pressure.
Vital sign measurements will be performed within ±10 minutes of the scheduled time
points
8.5.3 12-Lead ECGs
12-Lead ECGs will be recorded after 5 minutes supine rest on the Day of screening
visit, Day 0, Day 14±1, Day 28 and at follow up (See Appendix B). The 12-lead
ECGs will be recorded in triplicate at each time point, with each ECG separated by
approximately 1 minute at each time point.
8.5.4 Physical examinations
A full physical examination will be performed on the day of screening visit.
Abbreviated Physical examination will be done on Day 0, Day 28 and Follow-up.
8.5.5 Chest X ray
Chest X ray will be performed only in India and on the day of screening to exclude
pulmonary TB-infection.
8.5.6 Laboratory investigations and bioanalysis of blood samples
The sampling schedule for laboratory assessments are as mentioned in Appendix B.
The Safety laboratory investigations will be done at the local lab of the respective
study centre. The analysis of metabolic biomarkers (mentioned in Appendix A and B),
endocrinology assessments of all 40 Subjects will be done at Academic Medical
Centre, The Netherlands. Also, bioanalysis of blood samples for the determination of
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glucose and glycerol enrichment of all 40 Subjects will be done at Advanced
Laboratory of Endocrinology, AMC, Meibergdreef 9, room F2-131.3, 1100 DD
Amsterdam, The Netherlands. Blood samples collected from Study subjects at Veeda
Clinical Research Ahmedabad for evaluation of the parameters mentioned above will
be sent to AMC, The Netherlands.
Blood sampling
Blood sampling will be done for glucose monitoring (for maintaining Euglycemia) at
every 10 minutes. A window of ±1 minute will be allowed. Blood samples will be
taken at the following time points relative to the start of continuous glucose infusion
(taken as t=0): T= -2:00, -0:05, -0:00, 0:05, 1:50, 1:55, 2:00, 2:05, 2:10, 4:00, 4:05,
4:10, 4:15, 4:20 (14 samples during one clamp procedure).
The actual time of blood sampling will be recorded in the CRF. At each time point,
the blood samples will be collected into the appropriate polypropylene tube. The tubes
will be inverted gently 8 to 10 times before processing. The tubes will be kept in an
ice bath before centrifugation. Within 30 minutes of blood sample collection, the
samples will be centrifuged at approximately 3000 g for 10 minutes at 4ºC. The
plasma obtained from each sample will then be transferred into four labeled
polypropylene tubes and frozen immediately. The tubes will be stored at a
temperature below −70ºC prior to shipment. One set of samples will be sent to the
Advanced Laboratory of Endocrinology, AMC the Netherlands for bioanalysis of
each d2-glucose and d5-glycerol, insulin, cortisol, glucagon, catecholamines, other
biomarkers and as duplicate and back-up. All safety laboratory investigations will be
done at the local lab of the respective study centre.
Shipment
The plasma samples for bioanalysis will be shipped to the following bioanalytical
facility on dry ice, with a portable data logger (sample shipment log):
Laboratory of Special Endocrinology, AMC
Meibergdreef 9, room F2-131.3
1100 DD Amsterdam, The Netherlands
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The samples will be checked by the bioanalytical facilities as soon as possible after
they are received, and receipt will be documented.
8.5.7 Bioanalysis
Plasma samples will be analysed using a validated GCMS method.
8.5.8 Sample retention / destruction
The plasma samples will be retained by the bioanalytical facilities for 1 month after
the final study report has been signed off. The samples will be destroyed/returned
back to sponsor after obtaining written consent from the sponsor for the same.
Duplicate Samples will be sent to the bioanalytical facility after the confirmation of
receipt of first set is provided to the study centre.
8.6 Appropriateness of Measurements
All planned assessments are standard measurements for this type of study and are
considered appropriate.
8.7 Total Volume of Blood
Table 1: Summary of Blood Volumes for Each Subject
Requirement Number and Volume of Samples Total
Clinical laboratory
tests
Hematology,
endocrinology
20 x 4.5 ml 90
Haemostasis 5 x 2.7 ml 13.5
Biochemistry 5 x 4.5 ml 22.5
Viral Serology 1 x 10 ml 10
SIRT(for subjects
at Veeda CR only)
3 x10 ml 30
Clamp Day
(including baseline
measurements,
reserve samples
and glucose during
clamp)
2 x (125.1ml+ 20x 0.5ml
(glucose))
270.2
Total
For subject at AMC
For subject at Veeda CR
406.2ml
436.2 ml
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8.8 Withdrawal Criteria
Subjects may decide to withdraw from the study at any time without prejudice to their
further medical care. Although a subject is not obliged to give his reason(s) for
withdrawing prematurely from a trial, the investigator should make a reasonable effort
to ascertain the reason(s) while fully respecting the subject’s rights. The investigator
may withdraw a subject for any of the following reasons:
• adverse event: if subject is unwilling to continue because of an AE or if
continued participation of the subject would be an unnecessary risk to the
subject’s health, in the opinion of the investigator
• non-compliance
• protocol deviation
• lost to follow-up
• study blind is broken
• sponsor request
• following review of safety/tolerability data
• other.
8.9 Withdrawal Procedures
If a subject withdraws from the study, the primary reason for withdrawal must be
recorded in the CRF. The procedures scheduled for early withdrawal should be
performed if possible, as well as any other additional procedures requested by the
investigator for safety purposes. The last date of study drug administration must be
documented. Appropriate follow-up of withdrawn subjects will be performed, as
required. Attempts to contact a subject who withdraws from a study must be
documented.
8.10 Replacement of Dropout/Withdrawals
Replacement of dropout/withdrawals will be done by Pharmacy personnel at the
CRU, who is unblind to the study drug. Pharmacist at the CRU will manage any
dropout/withdrawal by providing different subject ID and randomisation code to the
reserve subject but will receive same drug (active treatment/placebo) as allotted to the
replaced subject. For example, if subject 002 is withdrawn, then the first replacement
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will be allocated with randomisation code A002, if second replacement of same
subject happen then B002, for third replacement C002.
8.11 Follow up of subjects withdrawn from treatment
Should a subject request or decide to withdraw from the study, all efforts will be made
to complete and report the observations as thoroughly as possible up to the date of
withdrawal, and for subjects who are withdrawn, the follow-up assessments should be
performed within 8-10 days after the last drug administration.
8.12 Study Termination
The sponsor, investigator or Institutional Review Board (IRB)/Independent Ethics
Committee (IEC) may terminate the study at any time if any of the following criteria
are met.
• There is new information about the study drug that indicates the risk/benefit
profile is no longer acceptable for the study to continue.
• There is a significant deviation from the protocol or violation of Good Clinical
Practice (GCP) that compromises the study results, subject safety or ability to
address the study objectives.
If a trial is prematurely terminated or suspended for any reason, the investigator
should promptly inform the trial subjects and should assure appropriate therapy
and follow-up. The sponsor, IRB/IEC and regulatory authority (ies) should be
notified
9. SAFETY REPORTING
9.1 Safety and tolerability assessments and reporting
9.1.1 Safety Monitoring
In this study with small sample size and short duration, the independent physician
will perform safety control by reviewing clinical safety parameters throughout the
conduct of the study. Clinical safety parameters will include laboratory analysis,
urine analysis and ECG
9.1.2 Section 10 WMO event
In accordance to section 10, subsection 1, of the WMO, the investigator will inform
the subjects and the reviewing accredited IEC if anything occurs, on the basis of
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which it appears that the disadvantages of participation may be significantly greater
than was foreseen in the research proposal. The study will be suspended pending
further review by the accredited IEC, except insofar as suspension would jeopardise
the subjects’ health. The investigator will take care that all subjects are kept informed.
9.1.3 Adverse events and serious adverse events
Adverse events are defined as any undesirable experience occurring to a subject
during a clinical trial, whether or not considered related to the investigational drug.
All adverse events reported spontaneously by the subject or observed by the investiga-
tor or his staff will be recorded. A serious adverse event is any untoward medical
occurrence or effect that at any dose
1. results in death;
2. is life threatening (at the time of the event);
3. requires hospitalisation or prolongation of existing inpatients’ hospitalisation;
4. results in persistent or significant disability or incapacity;
5. is a congenital anomaly or birth defect;
6. is a new event of the trial likely to affect the safety of the subjects, such as an
unexpected outcome of an adverse reaction, lack of efficacy of an IMP used
for the treatment of a life threatening disease, major safety finding from a
newly completed animal study, etc.
All SAEs will be reported to the accredited IEC that approved the protocol,
according to the requirements of that IEC.
9.1.4 Reporting of adverse event
For each subject, all AEs will be collected from the time of first dose administration
through to the post-study follow-up visit. On arrival at the trial unit, at regular
intervals during study, and at follow up, each subject will be asked a non-leading
question such as “How have you been feeling since last asked?” Any AEs reported in
response to questioning, as well as AEs reported spontaneously and occurring at any
other time after dosing, will be recorded in the subject’s CRF. For each AE, the
following information will be recorded:
• AE description
• start and stop dates and times
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• single episode or intermittent
• severity: mild (easily tolerated), moderate (interferes with daily activities) or
• severe (prevents normal daily activities)
• relationship to study drug (definite, probable, possible, unlikely or not
related)*
• outcome (resolved, resolved with sequelae, or ongoing)
• whether serious or not.
* Definitions for the relationship to study drug assessment, to be made by the
investigator, are as follows:
Definite: The AE follows a reasonable temporal sequence from administration
of the drug, abates on withdrawal of study drug and re-appears upon
re-administration (rechallenge).
Probable: The AE follows a reasonable temporal sequence from the
administration of the drug, abates on withdrawal of study drug and
cannot be reasonably explained by the known characteristics of the
subject's clinical state or other factors, such as concomitant medication.
Possible: The AE follows a reasonable temporal sequence from the
administration of the drug but could have been produced by the
subject's clinical state or other factors, such as study procedures or
concomitant medication.
Unlikely: The temporal association between the AE and the drug is such that the
drug is unlikely to have any reasonable association with the AE.
Not related: The AE does not follow a reasonable temporal sequence from the
administration of the drug or the AE can be reasonably explained by
other factors, such as concomitant medication.
9.1.5 Procedures for reporting SAEs
All SAEs should be reported immediately to the sponsor. The immediate reports
should be followed promptly by detailed, written reports. The immediate and follow-
up reports should identify a subject by his initials and the subject number, and not by
personal information such as name or address. The investigator should also comply
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with the applicable regulatory requirement(s) related to the reporting of unexpected
serious adverse drug reactions to the regulatory authority(ies) and the IRB/IEC.
9.1.6 Emergency procedures
The subject should be given supportive treatment depending on the symptoms.
9.1.7 Suspected unexpected serious adverse reactions (SUSAR)
Adverse reactions are all untoward and unintended responses to an investigational
product related to any dose administered. Unexpected adverse reactions are adverse
reactions, of which the nature, or severity, is not consistent with the applicable
product information (e.g. Investigator’s Brochure for an unapproved IMP or Summary
of Product Characteristics (SPC) for an authorised medicinal product). The sponsor
will report expedited the following SUSARs to the IEC: SUSARs that have arisen in
the clinical trial that was assessed by the IEC; SUSARs that have arisen in other
clinical trial of the same sponsor and with the same medicinal product, and that could
have consequences for the safety of the subjects involved in the clinical trial that was
assessed by the IEC. The remaining SUSARs will be recorded in an overview list
(line-listing) that will be submitted once every half year to the IEC. This line-listing
provides an overview of all SUSARs from the study medicine, accompanied by a
brief report highlighting the main points of concern. The sponsor will report expedited
all SUSARs to the competent authority, the Medicine Evaluation Board and the
competent authorities in other Member States. The expedited reporting will occur not
later than 15 days after the sponsor has first knowledge of the adverse reactions. For
fatal or life threatening cases the term will be maximal 7 days for a preliminary report
with another 8 days for completion of the report.
9.1.8 Annual safety report
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a
year throughout the clinical trial, a safety report to the accredited IEC’s, competent
authority, Medicine Evaluation Board and competent authorities of the concerned
Member States. This safety report consists of:
• a list of all suspected (unexpected or expected) serious adverse reactions, along
with an aggregated summary table of all reported serious adverse reactions, ordered
by organ system, per study;
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• a report concerning the safety of the subjects, consisting of a complete safety
analysis and an evaluation of the balance between the efficacy and the harmfulness of
the medicine under investigation.
9.1.9 Follow-up of adverse events
All adverse events will be followed until they have abated, or until a stable situation
has been reached. Depending on the event, follow up may require additional tests or
medical procedures as indicated, and/or referral to the general physician or a medical
specialist.
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10. DATA MANAGEMENT AND STATISTICAL ANALYSIS
10.1 Sample Size Calculation
It is intended that 40 subjects (20 subjects in India and other 20 in Amsterdam,
Netherlands) will complete the study. No formal sample size calculation has been
performed. We calculated that a sample size of 20 in each group will have 80% power
to detect an absolute difference in Rd of at least 15 µmol/kg·min (measure for
peripheral insulin sensitivity), before and after treatment, using a Wilcoxon (Mann-
Whitney) rank-sum test with a 0.05 two-sided significance level and assuming that the
common standard deviation is 15.
10.2 Data Handling
Data obtained in this study will be collected in eCRF prepared by the CRU of the
Academic Medical Centre, Amsterdam. The eCRF will be completed and monitored
in accordance with the principles of GCP. Source data is defined as all information in
original records and certified copies of original records of clinical findings,
observations, or other activities in a clinical trial necessary for the reconstruction and
evaluation of the trial. Source documents are original documents, data and records,
such as laboratory printouts, 12-lead ECG reports, dispensing records, and subject
files. Data recorded directly into the eCRFs will be considered as source. Full details
of procedures for data handling will be documented in the Data Management Plan.
AEs, medical history, and pre-existing (concurrent) conditions will be coded using the
Medical Dictionary for Regulatory Activities (MedDRA). Drugs will be coded using
the World Health Organization (WHO) Drug Dictionary.
10.3 Statistical Analysis
10.3.1 Descriptive statistics
Demographic and baseline characteristics will be summarized using descriptive
statistics for each group.
10.3.2 Univariate analysis
Because of the small sample size non parametric tests will be used to compare results
between the two treatment groups before and after treatment. Actual and percentage
change from baseline will be presented. All results are expressed as means and
standard deviations when the results are normally distributed or as median and range
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when they are not normally distributed. The data will be analyzed using SPSS,
version 16.
10.4 Safety and Tolerability Analysis
Safety and tolerability data will be summarised using the Safety Set, broken down by
dose level and for subjects on active treatment and on placebo. All AEs will be coded
using MedDRA. Data will be summarised using preferred term and primary system
organ class. Only treatment-emergent AEs, being events with an onset at or after the
first administration of study drug, will be presented in summary tables. Where
changes in severity are recorded in the CRF, the most severe incidence of the AE will
be reported in the tables. Rates will be calculated as the proportion of the number of
subjects with at least one AE related to the number of subjects treated. Frequency
tables indicating seriousness, severity, drug relation and the number of subjects will
be presented. Summary tables will present descriptive statistics for biochemistry and
haematology parameters, corrected QT (QTc) interval, diastolic and systolic blood
pressure and heart rate measurements. Frequency tables will be presented for physical
examination findings and urinalysis. Qualitative changes in laboratory values will be
presented by shift tables. No significance testing will be performed on safety and
tolerability data.
11. ETHICAL AND REGULATORY CONSIDERATIONS
11.1 Regulation statement
This study will be conducted in accordance with the protocol, the ethical principles
that have their origin in the Declaration of Helsinki, and that are consistent with the
principles of the International Conference on Harmonisation (ICH) (Step 5) 'Guidance
on Good Clinical Practice', ICMR guidelines, and with procedures oriented to Good
Laboratory Practice, and the applicable regulatory requirement(s).
11.2 Recruitment and consent
Subjects will be recruited from the outpatient’s clinic of AMC or from local
advertisement or from pre-existing database of the study centre. A study-specific
subject information sheet and consent form for each part of the study will be prepared
in accordance with the principles of GCP, the Declaration of Helsinki and all
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applicable laws and regulations. The information sheet will explain clearly the nature
of the study and study drug, including the study objectives, the potential risks and
benefits, and the procedures involved for the subject if he was to take part. For each
individual volunteer, the investigator or designee will provide a detailed explanation
of the study, both verbally and with the use of the written subject information sheet. If
the subject decides to participate in the study, the informed consent form must be
signed and dated by the subject and the investigator (or designee who conducted the
informed consent discussion). The original signed informed consent form must be
stored in the trial master file at the trial unit. The date that informed consent was
provided must be recorded in the subject’s CRF. A copy of the signed informed
consent form and the information sheet must be given to the subject. The study-
specific informed consent form must be signed before any protocol-related procedures
are performed. The subject information sheet should be revised whenever important
new information becomes available that may be relevant to the subject’s consent. Any
revised written information and consent form should receive the IEC’s approval prior
to use.
11.3 Benefits and risks assessment, group relatedness
This study does not have specific advantages for the study subjects. The results of this
study may help researchers learn whether TRC150094 may be beneficial for the
treatment of male subjects with the metabolic syndrome. The most important known
adverse event of TRC150094 is headache. [6,6-2H2] glucose is glucose labelled with
a stable isotope of hydrogen, which behaves as the natural substrate and has no side
effects. [1,1,2,3,3-2H2] labelled glycerol is glycerol labelled with 5 stable isotope of
hydrogen, which behaves as the natural substrate and has no side effects. Actrapid is
fast acting insulin, a hormone that could induce hypoglycemia. However, it is not in
the scope of this protocol to allow hypoglycemia to occur, since plasma glucose
concentration will be fixed at 5 mmol/l by a variable infusion of glucose 20% guided
by frequent bedside glucose measurements An overview of the blood samples taken
during the clamp is provided in Appendix B. The total volume of blood sample is
361.2.ml for AMC subjects and 391.2 ml for Indian subjects. This amount is not
considered to be of negative influence to the subject’s health. MR- spectroscopy of
the liver will be made which takes about 30 minutes. This spectroscopy is not
considered to be potentially harmful to subjects. An X-thorax will be performed
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during the screening visit, to exclude TB-infection, in patients in India only. The
radiation hazard of the X-thorax is 0.02 milliSievert per X-ray. For comparison:
background radiation is 2 milliSievert per year for every person.
Incentives: In the Netherlands patients will receive 100 euro for each study visit (not
screening), this includes reimbursement for travel costs.
11.4 Compensation for study related illness/injury
Subject will be treated and/or compensated for any study-related illness/injury
pursuant to the information provided in the Compensation for Injury section of the
Informed Consent. Compensation to study subjects will be as per the local regulatory
practices. Every subject is insured in accordance with the local laws against damage
to health which might occur during the conduct of study and the material damage
which occur in connection thereto. The subject insurance and travel insurance (if
appropriate) is taken by the Sponsor.
12. ADMINISTRATIVE ASPECTS AND PUBLICATION
12.1 Handling and storage of data and documents
The investigator should maintain a list of appropriately qualified persons to whom
he/she has delegated trial duties. All persons authorized to make entries and/or
corrections on eCRFs will be included on the Authority Form. The investigator must
ensure that the subject’s confidentiality is maintained. On the eCRFs or other
documents subjects should be identified by subject ID and randomization number
only. The Investigator is obligated to inform and obtain the consent of the subject to
permit named representatives to have access to his/her study-related records without
violating the confidentiality of the subject. Source documents are the original
documents, data, and records from which the subject’s eCRF data are obtained. These
include but are not limited to hospital records, clinical and office charts, laboratory
and pharmacy records, diaries, microfiches, radiographs, and correspondence.
The Investigator and study staff are responsible for maintaining a comprehensive and
centralized filing system of all study-related (essential) documentation, suitable for
inspection at any time by representatives from the Sponsor or designee, and/or
applicable regulatory authorities. Elements should include:
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• Subject files containing informed consents, and supporting copies of source
documentation
• Study files containing the protocol with all amendments, Investigator’s Brochure,
copies of pre-study documentation, and all correspondence to and from the IEC as
well as to and from the Sponsor or designee.
• Study files containing documentation of study drug receipt, accountability, return,
and all drug related correspondence In addition, all source documents supporting
entries in the eCRFs must be maintained and be readily available. No study document
should be destroyed without prior written agreement between the Sponsor or designee
and the Investigator. Should the Investigator wish to assign the study records to
another party or move them to another location, he/she must notify the Sponsor or
designee in writing of the new responsible person and/or the new location. The
Sponsor representative or designee and regulatory authority inspectors are responsible
for contacting and visiting the Investigator for the purpose of inspecting the facilities
and, upon request, inspecting the various records of the trial (for example, eCRFs and
other pertinent data) provided that subject confidentiality is respected. The Sponsor
representative or designee is responsible for inspecting the eCRFs at regular intervals
throughout the study to verify adherence to the protocol; completeness, accuracy, and
consistency of the data; and adherence to local regulations on the conduct of clinical
research. The monitor should have access to subject medical records and other study-
related records needed to verify the entries on the eCRFs. In accordance with ICH
GCP and the Sponsor’s audit plans, this study may be selected for audit by
representatives from the Sponsor’s Clinical and Quality Assurance Department (or
designees). Inspection of study center facilities (e.g., pharmacy, drug storage areas,
laboratories) and review of study-related records will occur to evaluate the trial
conduct and compliance with the protocol, ICH GCP, and applicable regulatory
requirements.
12.2 Amendments
Amendments are changes made to the research after a favourable opinion by the
accredited IEC has been given. All amendments will be notified to the IEC that gave a
favourable opinion. A ‘substantial amendment’ is defined as an amendment to the
terms of the IEC application, or to the protocol or any other supporting
documentation, that is likely to affect to a significant degree:
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• the safety or physical or mental integrity of the subjects of the trial;
• the scientific value of the trial;
• the conduct or management of the trial; or
• the quality or safety of any intervention used in the trial.
All substantial amendments will be notified to the IEC and to the competent authority.
Non-substantial amendments will not be notified to the accredited IEC and the
competent authority, but will be recorded and filed by the sponsor.
12.3 Annual progress report
The sponsor/investigator will submit a summary of the progress of the trial to the
accredited IEC once a year. Information will be provided on the date of inclusion of
the first subject, numbers of subjects included and numbers of subjects that have
completed the trial, serious adverse events/ serious adverse reactions, other problems,
and amendments.
12.4 End of study report
The sponsor will notify the accredited IEC and the competent authority of the end of
the study within a period of 90 days. The end of the study is defined as the subject’s
last visit. In case the study is ended prematurely, the sponsor will notify the accredited
IEC and the competent authority within 15 days, including the reasons for the
premature termination. Within one year after the end of the study, the
investigator/sponsor will submit a final study report with the results of the study,
including any publications/abstracts of the study, to the accredited IEC and the
CompetentAuthority.
12.5 Public disclosure and publication policy
Upon completion of the trial (or early termination), the investigator should provide
the IEC with a summary of the trial’s outcome, and the regulatory authority(ies) with
any reports required. A clinical study report will be prepared in accordance with the
ICH E3 guideline: Note for guidance on structure and content of clinical study reports
(CPMP/ICH/137/95). The detailed obligations regarding the publication of data or
information from this study will be set out in a separate agreement between the
investigator and sponsor. The identity of individual subjects will not be revealed in
any reports or publications.
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13. QUALITY CONTROL AND QUALITY ASSURANCE
The following activities will be undertaken to ensure the quality of trial-related
activities:
• adherence to the trial site standard operating procedures to maintain accurate and
consistent practices and procedures
• conduct of a site initiation visit to ensure the investigator and all personnel
involved in the trial understand the protocol, including the study procedures and
their responsibilities
• completion of eCRF in accordance with GCP requirements to ensure accurate and
reliable data
• periodic monitoring to ensure the trial data are accurate, complete and verifiable
from source documents, and that the protocol is being followed
• data entry of eCRF data and data management of all trial data will be performed in
accordance with GCP requirements.
The trial site may be subject to quality assurance audits by the sponsor or its designee.
If an audit is undertaken, the site will be contacted in advance to arrange an auditing
visit.
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APPENDIX A: Clinical Laboratory Tests
Haematology
Red blood cells
Total Leukocytes count (cell count)
Differential WBC count (absolute and
%) including neutrophils, eosinophils,
basophils, monocytes, lymphocytes
Haemoglobin
Platelets (cell count)
Packed cell volume
ESR
Biochemistry
Total Protein
Albumin
Urea (BUN)
Creatinine
Fasting Blood Glucose & Insulin
Electrolytes: Serum Na, K, HCO3,Ca, P,
Cl-
Liver Function Tests: ALT, AST,
Bilirubin (Total and Direct)
LDH, GGT
Haemostasis
Prothrombin time,
aPTT (Activated
Partial thromboplastin time),
Fibrinogen.
Endocrinology
Thyroid Profile (T3, T4, fT4, TSH)
Metabolic markers:
FFA, Leptin, TNF-alpha, IL-6, IL-1beta,
IL-10, MCP-1, Serum Glucagon, Serum
Epinephrine, Resistin, Adiponectin,
Apolipoproteins, CRP, Other metabolic
markers (Refer Appendix B).
Urinalysis
pH, specific gravity, ketones,
protein,glucose, urobilinogen, bilirubin,
nitrite, erythrocytes, leukocytes
Lipid Parameters
Total cholesterol, TG,
LDL – Cholesterol, VLDL, HDL
Viral serology
HBsAg, hepatitis C antibody, HIV I,
HIV II
Drugs of abuse
amphetamines, barbiturates,
benzodiazepines, cannabinoids, cocaine,
opiates, methadone
Alcohol breath test
Time points for the clinical laboratory tests are mentioned in Appendix B.
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APPENDIX B: Study visits.
Day 0
During clamps
1 S
0 min 115
min
120
min
125
min Last 20 mins
(at 5 min intervals)
Last 20 mins
(at 5 min intervals)
Day
14
Day
28
Day
35
Invest T -2:00 -0:05 0:00 0:05 1:50 1:55 2:00 2:05 2:10 4:00 4:05 4:10 4:15 4:20
Informed
consent ×
Demographics,
smoking status ×
Height, weight,
BMI, Waist Circ × × × × ×
Sagittal
abdominal
diameter
× ×
Medical history;
pre-existing
conditions
×
Prior/concomita
nt medication
check
×
Physical
examination × × × ×
Vital signs × × × × ×
Body
temperature ×
12-Lead ECG × × × × ×
Chest X ray (In
India only) ×
Hematology × × × × ×
Safety
Biochemistry × × × × ×
Serology ×
Lipid profile × × × × ×
Urine Analysis × × × × ×
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Day 0
During clamps
2 S
0 min 115
min
120
min
125
min Last 20 mins
(at 5 min intervals)
Last 20 mins
(at 5 min intervals)
Day
14
Day
28
Day
35
Invest T -2:00 -0:05 0:00 0:05 1:50 1:55 2:00 2:05 2:10 4:00 4:05 4:10 4:15 4:20
D2 glucose × × × × × × × × × × × × × ×
D5 glycerol × × × × × × × × × × × × × ×
Serum insulin × × × × × × × × × ×
Serum glucagon × × ×
Catecholamines × × ×
cortisol × × ×
FFA × × ×
Leptin × × ×
TNF Alpha × × ×
IL-6 × × ×
IL-1beta × × ×
IL-10 × × ×
MCP-1 × × ×
Other Metabolic
markers (such as
Plasma SCD-1
activity, plasma
3-OHB,
osteopontin)#
× × ×
No
t a
pp
lica
ble
At
da
y 2
8 t
he
sam
e sc
hem
e a
s d
ay
0 w
ill
be
foll
ow
ed
No
t a
pp
lica
ble
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Dosing period: Day 1 to Day 28
Hepatic MRS will be done on Day 0 and Day 28.(A deviation of ±2 days is acceptable) #
These are exploratory investigations and the exact list will be determined based on clamp study findings. Till then, samples will be preserved.
Day 0
During clamps
3 S
0 min 115
min
120
min
125
min Last 20 mins
(at 5 min intervals)
Last 20 mins
(at 5 min intervals)
Day
14
Day
28
Day
35
Invest T -2:00 -0:05 0:00 0:05 1:50 1:55 2:00 2:05 2:10 4:00 4:05 4:10 4:15 4:20
Resistin × × ×
Adiponectin(Tot
al and HMW) × × ×
Apo A1 × × ×
Apo B × × ×
CRP × × ×
No
t a
pp
lica
ble
Sa
me
sch
eme
as
da
y 0
No
t a
pp
lica
ble
SIRT expression
(For Indian
Cohorts only) × × × ×
Drugs of abuse
(urine) ×
Alcohol breath
test ×
Randomisation ×
Compliance
check for Life
style
instructions &
drug
× × × ×
AE check and
change in
concomitant
medication
× × × ×
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APPENDIX C: Flow Chart of Clamp Procedure:
App Time Day Durati
on/ T= Activity
20:00 -1 and
27 13 hr Fasting
07:30 0 and 28 30 min MRS
08:00 0 and 28 -2hr
Two catheters will be inserted in both the arms. Right side
will be used for infusion and Left side will be used for blood
sample collection
09:00 0 and 28 -2 hr
• Blood drawing for background enrichment of plasma
glucose
• Priming dose: equivalent to 100 min of infusion at rate
0.11 µmol/kg per min
• A continuous infusion of [6,6-2H2]-glucose (>99%
enriched, Cambridge Isotopes, Massachusetts, USA) is
started at a rate of 0.11 µmol/kg per min till the end of
clamp (6:20hr).
• Priming dose: 1.6 µmol/kg/min. A continuous infusion
of [1,1,2,3,3-2H2]-glycerol (>99% enriched,
Cambridge Isotopes, Massachusetts, USA) is started at
a rate of 0.11 µmol/kg per min till the end of clamp
(5:20hr).
10:50 0 and 28 -0:10
10:55 0 and 28 -0:05
11:00
0 and
28 0:00
Blood sample will be collected for determination of
• Glucose and glycerol enrichment
• Glucoregulatory hormones (Insulin, Glucagon &
Catecholamine)
• FFA
11:00
0 and 28
0:00
The hyperinsulinemic clamp will start with a continuous
infusion of insulin (Actrapid 100U/ml, Novo Nordisk) for 2h
at a rate of 20mU/m2 body surface area per min.
11:00-13:05
BG will be
estimated at
every 10 min
0 and 28
0:00-
2:05
Plasma glucose is measured every 10 min (via Biosen/YSI
analyzer) and glucose 20% is infused at a variable rate to
maintain plasma glucose at 5.0 mmol/l. [6,6-2H2]-glucose is
added to the 20% glucose solution to achieve glucose
enrichments of 1% to minimize changes in isotopic
enrichment due to changes in the infusion rate of exogenous
glucose, and thus to allow for accurate quantification of
endogenous glucose production and uptake
12:50 1:50
12:55 1:55
13:00 2:00
13:05 2:05
13:10
0 and 28
2:10
Blood sample will be collected for determination of
• Glucose and glycerol enrichment
• Glucoregulatory hormones Insulin, Glucagon &
Catecholamine)
• FFA
13:15– 15:15 0 and 28 2:15-
4:15
The infusion of insulin (Actrapid 100U/ml, Novo Nordisk) is
increased to a rate of 60mU/m2 body surface area per min.
13:15-15:15
BG will be
0 and 28 2:15-
4:15
Plasma glucose is measured every 10 min (via Biosen/YSI
analyzer) and glucose 20% is infused at a variable rate to
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estimated at
every 10 min
maintain plasma glucose at 5.0 mmol/l. [6,6-2H2]-glucose is
added to the 20% glucose solution to achieve glucose
enrichments of 1% to minimize changes in isotopic
enrichment due to changes in the infusion rate of exogenous
glucose, and thus to allow for accurate quantification of
endogenous glucose production and uptake
15:00 4:00
15:05 4:05
15:10 4:10
15:15 4:15
15:20
0 and 28
4:20
During the last 20 min of this insulin infusion step, blood
samples are drawn at 5 min intervals for determination of
• Glucose and glycerol enrichment,
• Glucoregulatory hormones
• FFA
15:20 0 and 28 4:20 Stop insulin infusion & Double the glucose infusion rate
15:20 0 and 28 4:20 Provide a carbohydrate rich meal to volunteer.
15:20 onwards
BG will be
estimated at
every 10 min
0 and 28
4:30
If plasma glucose reading in > 7 mmol/l (>126 mg/dl), then
reduce the glucose infusion rate to half, until it becomes less
than 5 ml/hr.
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